Compositions and methods for protecting against airborne pathogens and irritants

ABSTRACT

The present disclosure features methods and compositions for enhancing the ability of the respiratory membranes to filter airborne pathogens and protect a subject from respiratory infections that result from inhalation of such pathogens. In particular, the disclosure provides antimicrobial compositions that prevent and treat respiratory infections caused by bacteria, fungi, and viruses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. application Ser. No.15/442,604, filed Feb. 24, 2017, which claims priority to U.S. App. No.62/299,755, filed Feb. 25, 2016, the entire contents of which are herebyincorporated by reference.

FIELD OF THE DISCLOSURE

The disclosure relates to compositions and methods of augmenting thehealth and filtering capabilities of epithelial and mucous membranes byenhancing the integrity of their natural, protective secretions. Inparticular, the disclosure relates to compositions and methods forprotecting the epithelial and mucous membranes of a subject frominfection by airborne pathogens, such as viruses, bacteria, and fungi,and irritation from undesirable airborne particles such as allergens,irritants, or odorants. The disclosure further relates to compositionsfor application to the respiratory tract (e.g., the nasal and oralmucosa) of a human for prophylaxis of microbial and viral infections,particularly human rhinovirus (HRV) and human influenza virusinfections.

BACKGROUND

Respiratory infections typically occur when airborne pathogens come intocontact with mucous membranes (e.g., nasal membranes, nasal hairs,esophageal membranes, and the like) via inhaled or ingested liquid oraerosol droplets. Inhalation or ingestion of pathogens through the noseor mouth is a primary cause of respiratory disease and may also causesystemic disease such as poliomyelitis or foot and mouth disease.Airborne pathogens may enter the lungs after inhalation or ingestion, orthey may bind receptors found on nasal and other membranes throughoutthe upper and lower respiratory tracts which serve as an entry points bywhich pathogens, allergens, or irritants can enter into the bloodstreamand cause respiratory, as well as other, types of infection or allergicreaction. Unfortunately, there is no convenient, effective way tominimize or prevent infection or allergy by inhaled or ingestedmicroorganisms. Therefore, there is an urgent need to develop newcompositions and methods to protect against airborne pathogens,allergens, and irritants, and particularly against viruses, particularlyhuman rhinovirus (HRV), human influenza virus, or both.

SUMMARY OF INVENTION

In accordance with the foregoing objectives and others, the presentdisclosure features compositions, such as nasal sprays, oral sprays,oral rinses, lozenges, and the like, and associated methods of usingsuch compositions for enhancing the ability of the epithelial membranesto filter certain airborne pathogens. In particular, the disclosureprovides antimicrobial compositions that prevent and treat respiratoryinfections and allergies caused by irritants, allergens, bacteria,fungi, and viruses. In preferred implementations, the compositionsprotect a subject from viral infections, particularly from humanrhinovirus and/or human influenza virus.

In one aspect of the invention, a composition is provided forprophylaxis or treatment of a human subject suffering from, or at riskof suffering from, a respiratory infection. The composition may compriseone or more antimicrobial or antiviral compounds dispersed in a carrier,typically, but not necessarily, a liquid carrier. The liquid carrier isideally, but not necessarily, of suitable rheology to be sprayed as anaerosol or fine mist. The composition may comprise one or moreingredients selected from the group consisting of an emollient, anocclusive, a humectant, a carrier, an excipient, an emulsifier, and anessential oil. In some embodiments, the composition for prophylaxis ortreatment of respiratory infection may comprise active ingredient thatcombat infection against viruses that bind intercellular adhesionmolecule 1 (ICAM-1) and/or viruses that bind sialic acid (orextracellular portions thereof). In one implementation, a composition isprovided for prophylaxis or treatment of a human subject suffering from,or at risk of suffering from, infection of the respiratory tract withhuman rhinovirus (HRV) comprising, in a suitable liquid carrier: (i)soluble ICAM-1 (“sICAM-1”) and/or an ICAM-1 inhibitor; (ii) lysozyme;and (iii) lactoferrin (e.g., apolactoferrin). In another implementation,a composition is provided for prophylaxis or treatment of a humansubject suffering from, or at risk of suffering from, infection of therespiratory tract with human influenza virus comprising, in a suitableliquid carrier: (i) sialic acid (e.g., sialyllactose); (ii) lysozyme;(iii) lactoferrin; and (iv) optionally, a neuraminidase inhibitor suchas, for example, quercetin. In yet another implementation, a compositionis provided for prophylaxis or treatment of a human subject sufferingfrom, or at risk of suffering from, infection of the respiratory tractwith human rhinovirus (HRV) and human influenza virus comprising, in asuitable liquid carrier: (i) soluble ICAM-1 (sICAM-1) and/or an ICAM-1inhibitor; (ii) lysozyme; (iii) lactoferrin, (iv) sialic acid and/or aderivate therefore (e.g., sialyllactose); and (v) optionally, aneuraminidase inhibitor. Any of the compositions according to theseembodiments, may further comprise one or more of zinc peroxide, copper,and silver. Any of the compositions according to these embodiments, mayfurther comprise carrageenan. Any of the compositions according to theseembodiments, may further comprise one or more of IgA, IgG, and IgM. Thecompositions may further comprise one or more ingredients selected fromthe group consisting of marshmallow extract, Calendula extract, citruspeel extract, honey extract, rosemary extract, myrrh extract,Helichrysum extract, arrowroot extract, neem oil, vitamin C, vitamin E,and grapefruit seed extract. The carrier may be aqueous, and may includeone or more pharmaceutically acceptable excipients, including, withoutlimitation, diluents, buffering agents, pH adjusters (e.g., citric acid,etc.), thickeners and suspending agents (e.g., gum acacia, xanthan gum,hydroxypropylmethylcellulose, microcrystalline cellulose, sodiumcarboxymethylcellulose, etc.), rheology modifiers, preservatives (e.g.,phenethyl alcohol, benzalkonium chloride, sodium EDTA, etc.),isotonicity adjusters (e.g., sodium chloride, polyols, sucrose, etc.),humectants (e.g., glycerin), surfactants (e.g., polysorbates, such aspolysorbate 80, sucrose palmitate, glyceryl stearate, glyceryl stearatecitrate, acetylated hydrogenated vegetable glyceride, etc.), and tastemodifiers, to name a few. Any excipients should be compatible with thehuman mucosa and epithelium, and should not cause excessive drying orirritation to the mucosa or epithelium. The excipients should alsoaccount for the fact that water will tend to evaporate at bodytemperature and as such a secondary solvent may be included to aid inmaintaining the soluble components in solution. The carrier may includea polyol, such as a C2-C polyol, including without limitation, glycerin,propylene glycol, 1,3-propane diol, butylene glycol, 1,4-butane diol,erythritol, threitol, arabitol, xylitol, mannitol, sorbitol, pentyleneglycol, hexylene glycol, caprylyl glycol, hydrogenated starchhydrolysates, isomalt, maltitol, and the like. The compositions maycomprise an amount of an alcohol, such as ethanol, provided it is in anamount that does not irritate or dry the mucosa. In some embodiments,the compositions are free of ethanol. In one embodiment, the carrier isan aqueous carrier including from about 1-95% or from about 5-50% orfrom about 10-40% or from about 15-35% or from about 20-30%1,3-propanediol, on a (v/v), (w/v), or (w/w) basis. In some embodiments,the composition may have a kinematic viscosity ranging from about1-1,500 or from about 5-1,000 or from about 10-750 or from about 20-500centiStokes (mm²/s). The compositions may have a Newtonian ornon-Newtonian rheology. The compositions may be, for example, shearthinning and/or thixotropic, such that they readily flow through a spraynozzle and form a mist of suitable droplet size on shearing, but thickenin situ to form a film on the mucosa which is resistant to clearancefrom the nasal or oral cavity such that the active remain on the mucosafor a time sufficient to neutralize pathogens in contact with themucosa. Typically, the composition will be of suitable viscosity topossess a residence time on the mucosa of the nasal or oral cavities ofat least 1 minute, more preferably, at least 5, 10, 15, 20, 25, or 30minutes following application. The composition should be semi-permeablein order to permit virions and other pathogens to penetrate the film andcome into contact with the active ingredients, while possessing asufficient barrier function to inhibit evaporation of water and volatilesolvents in order to maintain the actives in solution.

In some embodiments, the composition may comprise:

-   -   (i) about 0.00000001%-10% by weight ICAM-1 (e.g., soluble        ICAM-1);    -   (ii) 0% (or from about 0.00000001%) to about 10% by weight of a        neuraminidase inhibitor;    -   (iii) about 0.00000001% to about 10% by weight of sialic acid        (e.g., sialyllactose, 2,3′-sialyllactose, and/or 2,6′        sialyllactose);    -   (iv) about 0.00000001% to about 10% by weight of lysozyme; and    -   (v) about 0.00000001% to about 10% by weight of lactoferrin        (e.g., apolactoferrin);        a pharmaceutically acceptable carrier and, optionally, one or        more excipients.

In some embodiments, the composition may comprise:

-   -   (i) about 0.000001%-1% (or to about 0.1%) by weight ICAM-1        (e.g., soluble ICAM-1); and/or    -   (ii) 0% (or from about 0.000001%) to about 1% (or to about 0.1%)        by weight of a neuraminidase inhibitor; and/or    -   (iii) about 0.000001% to about 0.001% (or to about 0.01%) by        weight of sialic acid (e.g., sialyllactose, 2,3′-sialyllactose,        and/or 2,6′ sialyllactose); and/or    -   (iv) about 0.0001% to about 5% (or to about 1%) by weight of        lysozyme; and/or    -   (v) about 0.00005% to about 5% (or to about 0.5%) by weight of        lactoferrin (e.g., apolactoferrin);        a pharmaceutically acceptable carrier and, optionally, one or        more excipients.

In some embodiments, the composition may comprise:

-   -   (i) about 0.0005%-0.05% by weight ICAM-1 (e.g., soluble ICAM-1);        and/or    -   (ii) 0% (or from about 0.005%) to about 0.05% by weight of a        neuraminidase inhibitor; and/or    -   (iii) about 0.000005% to about 0.05% by weight of sialic acid        (e.g., sialyllactose, 2,3′-sialyllactose, and/or 2,6′        sialyllactose); and/or    -   (iv) about 0.0025% to about 0.25% by weight of lysozyme; and/or    -   (v) about 0.00005% to about 0.1% by weight of lactoferrin (e.g.,        apolactoferrin);        a pharmaceutically acceptable carrier and, optionally, one or        more excipients.

The pharmaceutical composition may be used in a method of preventing ortreating respiratory infection. The respiratory infection may be due tohuman rhinovirus and/or human influenza virus. In some embodiments, acomposition for preventing or treating respiratory infection from humanrhinovirus (HRV) may comprise:

-   -   (i) about 0.00000001% to about 10% by weight soluble ICAM-1;    -   (ii) about 0.000005% to about 10% by weight lysozyme; and    -   (iii) about 0.00000025% to about 10% by weight lactoferrin;        and a pharmaceutically acceptable carrier and, optionally, one        or more excipients.

In some embodiments, a composition for preventing or treatingrespiratory infection from human rhinovirus (HRV) may comprise:

-   -   (i) about 0.000001%-1% (or to about 0.1%) by weight soluble        ICAM-1;    -   (ii) about 0.0001% to about 5% (or to about 1%) by weight        lysozyme; and    -   (iii) about 0.00005% to about 5% (or to about 0.5%) by weight        lactoferrin;        and a pharmaceutically acceptable carrier and, optionally, one        or more excipients.

In some embodiments, a composition for preventing or treatingrespiratory infection from human rhinovirus (HRV) may comprise:

-   -   (i) about 0.0005%-0.05% by weight soluble ICAM-1; and/or    -   (ii) about 0.0025% to about 0.25% by weight lysozyme; and/or    -   (iii) about 0.00005% to about 0.1% by weight lactoferrin;        and a pharmaceutically acceptable carrier and, optionally, one        or more excipients.

In some embodiments, a composition for preventing or treatingrespiratory infection from human influenza virus may comprise:

-   -   (i) about 0.0000001% to about 10% by weight of said sialic acid        (e.g., sialyllactose);    -   (ii) about 0.00000001% to about 10% by weight of said lysozyme;    -   (iii) about 0.00000001% to about 10% by weight of said        lactoferrin; and    -   (iv) 0% (or from about 0.00000001%) to about 10% by weight of a        neuraminidase inhibitor;        and a pharmaceutically acceptable carrier and, optionally, one        or more excipients.

In some embodiments, a composition for preventing or treatingrespiratory infection from human influenza virus may comprise:

-   -   (i) about 0.000005% to about 0.05% by weight of said sialic acid        (e.g., sialyllactose);    -   (ii) about 0.0001% to about 5% (or to about 1%) by weight of        said lysozyme;    -   (iii) about 0.00005% to about 5% (or to about 0.5%) by weight of        said lactoferrin; and    -   (v) 0% (or from about 0.01%) to about 10% by weight of a        neuraminidase inhibitor;        and a pharmaceutically acceptable carrier and, optionally, one        or more excipients.

In some embodiments, a composition for preventing or treatingrespiratory infection from human influenza virus may comprise:

-   -   (i) about 0.000005% to about 0.05% by weight of said sialic acid        (e.g., sialyllactose); and/or    -   (ii) about 0.0025% to about 0.25% by weight of said lysozyme;        and/or    -   (iii) about 0.00005% to about 0.1% by weight of said        lactoferrin; and/or    -   (vi) 0% (or from about 0.000001%) to about 1% (or to about 0.1%)        by weight of a neuraminidase inhibitor;        and a pharmaceutically acceptable carrier and, optionally, one        or more excipients.

In some embodiments, the compositions of the invention will be aqueoussolutions or suspensions comprising from about 0.5-5000 μg/mL (or fromabout 1-1000 μg/mL or from about 5-500 μg/mL) of lactoferrin (e.g.,apolactoferrin). In some embodiments, the compositions of the inventionwill be aqueous solutions or suspensions comprising from about0.25-10000 μg/mL (or from about 1-5000 μg/mL or from about 25-2500μg/mL) of lysozyme. In some embodiments, the compositions of theinvention will be aqueous solutions or suspensions comprising from about0.01-500 μg/mL (or from about 0.1-100 μg/mL or from about 0.5-50 μg/mL)of ICAM-1 (e.g, soluble ICAM-1). In some embodiments, the compositionsof the invention will be aqueous solutions or suspensions comprisingfrom about 0.01-2000 μg/mL (or from about 0.1-1000 g/mL or from about0.5-750 μg/mL) of sialic acid (e.g., sialyllactose). In someembodiments, the compositions of the invention will be aqueous solutionsor suspensions comprising from about 0.005-1000 μg/mL (or from about0.5-500 μg/mL or from about 0.25-375 μg/mL) of 3′ sialyllactose and/orfrom about 0.005-1000 μg/mL (or from about 0.5-500 μg/mL or from about0.25-375 μg/mL) of 6′ sialyllactose.

The pharmaceutical compositions according to the invention may be in theform of a nasal spray, nasal drops, oral spray, oral rinse, or lozenge.The carrier of the pharmaceutical composition may be selected to provideresidence time of the composition on the nasal and/or oral mucosa of atleast 1 minute, or at least 5 minutes, or at least 10 minutes, or atleast 15 minutes, or at least 20 minutes, or at least 25 minutes, or atleast 30 minutes following application. In some embodiments, thecomposition for application to the nasal or oral mucosa comprises one ormore antiviral and/or antimicrobial agents dispersed in a liquid carriercomprising from about 1-99% (v/v) water or from about 60-90% (v/v) waterand from about 10-40% (or from 20-30%) (v/v) of a polyol. In someembodiments, the pharmaceutically acceptable carrier is an aqueoussolution comprising from about 5-50% (v/v), or from about 10-40% (v/v),or from about 15-35% (v/v), or from about 20-30% (v/v) 1,3-propanediol.The composition may be capable of being sprayed or ingested onto themucosa, and is adapted to remain on the mucosa for at least 5 minutes(or at least 10 minutes, or at least 15 minutes, or at least 20 minutes,or at least 25 minutes, or at least 30 minutes) following applicationwithout substantially irritating or drying the mucosa.

Methods for prophylaxis and/or treatment of various viral infections areprovided. In some embodiments, the method for prophylaxis and/ortreatment of human rhinovirus infection, comprises applying any of thecomposition described herein to the nasal and/or oral mucosa of anindividual in need thereof. In some embodiments, the nasal and/or oralmucosa of individuals in need thereof has human rhinovirus in contacttherewith.

In one aspect, the invention provides for a pharmaceutical compositionfor preventing or treating subjects suffering from or at risk ofsuffering from a respiratory infection comprising: one or moreantimicrobial or antiviral compounds; and a base mixture comprising oneor more ingredients selected from the group consisting of a carrier, anemollient, an occlusive, a humectant, a polyol, an emulsifier, apreservative, a thickener or suspending agent, a surfactant, a pHadjuster, an isotonicity agent, and an essential oil. In an embodiment,the antimicrobial or antiviral compound is one or more selected from thegroup consisting of an antibody such as IgA, IgG, or IgM, a solubleICAM-1, an ICAM-1 inhibitor, sialic acid, a neuraminidase inhibitor,lactoferrin, a lysozyme, zinc, zinc compounds, silver, silver compounds,copper, copper compounds, and combinations thereof. In an embodiment,the neuraminidase inhibitor is selected from the group consisting ofquercetin, oseltamivir, zanamivir, laninamivir, and peramivir. In anembodiment, the ICAM-1 inhibitor is selected from the group consistingof an anti-ICAM-1 antibody, cytokine, CD11a, ezrin (EZR), CD18,glycyrrhetinic acid, pyrrolidinedithiocarbamate, NFkB activationinhibitor, heterocyclic thiazole, lipoic acid, efalizumab,4-[(4-Methylphenyl)thio]thieno[2,3-c]pyridine-2-carboxamide, silibinin,stilbenes, (+)-epigalloyl-catechin-gallate [(+)-EGCG], and combinationsthereof. In an embodiment, the one or more antimicrobial or antiviralcompounds include soluble ICAM-1 and sialic acid (e.g., sialyllactose,3′ sialyllactose, and/or 6′ sialyllactose). In an embodiment, the one ormore antimicrobial or antiviral compounds include lactoferrin, lysozyme,neuraminidase inhibitor, IgA, IgG, IgM, zinc peroxide (ZnO₂), copper,and silver. In an embodiment, the respiratory infection is selected fromthe group consisting of a rhinovirus infection, an influenza virusinfection, a fungal infection, and a bacterial infection. In anembodiment, one or more ingredients are selected from the groupconsisting of a marshmallow extract, a calendula extract, a citrus peelextract, a honey extracts, a rosemary extracts, a myrhh extract, aHelichrysum extract, a arrowroot extract, a neem oil, an argan oil, avitamin C, a vitamin E, a grapefruit seed extract, and combinationsthereof.

In one aspect, the invention provides for a method of prophylaxis ortreatment of respiratory infection in subjects suffering from or at riskof suffering from respiratory infection comprising: determining asubject is suffering from or at risk of suffering from a respiratoryinfection; and administering a composition according to the inventioncomprising one or more antimicrobial or antiviral compounds and a basemixture comprising one or more ingredients selected from the groupconsisting of a carrier, an emollient, an occlusive, a humectant, anemulsifier, and an essential oil. In an embodiment, the one or moreantimicrobial or antiviral compounds comprise soluble ICAM-1. In anembodiment, the one or more antimicrobial or antiviral compoundscomprise sialic acid or a derivative thereof (e.g., sialyllactose). Inone embodiment, the one or more antimicrobial or antiviral compoundscomprise lactoferrin (e.g., apolactoferrin). In one embodiment, the oneor more antimicrobial or antiviral compounds comprise lysozyme. In oneembodiment, the one or more antimicrobial or antiviral compoundscomprise a neuraminidase inhibitor. In one embodiment, the one or moreantimicrobial or antiviral compounds comprise IgA, IgG, and/or IgM. Inone embodiment, the one or more antimicrobial or antiviral compoundscomprise zinc peroxide (ZnO₂), copper, and/or silver. The compositionsmay be administered by any suitable route, including orally, topically,nasally, and combinations thereof. In an embodiment, the composition isadministered to nasal membranes. In an embodiment, the composition isadministered using a device selected from the group consisting of anatomizer, an inhaler, a nebulizer, a spray bottle, and a spray pump. Thecomposition may include a propellant or may be free of propellants.

These and other aspects of the invention will be better understood byreference to the following Detailed Description and appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the effect apolactoferrin treatment at 500 mcg/ml(HRV1-1), 50 mcg/ml (HRV1-2), and 5 mcg/ml (HRV1-3) on the integrity oftissue infected with Rhinovirus A16. TEER was monitored 24 (D1) and 48hours (D2) post-inoculation on MucilAir™ 3D media.

FIG. 2 illustrates the effect of lysozyme treatment at 2500 mcg/ml(HRV2-1), 250 mcg/ml (HRV2-2), and 25 mcg/ml (HRV2-3) on the integrityof tissue infected with Rhinovirus A16. TEER was monitored 24 (D1) and48 hours (D2) post-inoculation on MucilAir™ 3D media.

FIG. 3 illustrates the effect of soluble ICAM-1 treatment at 50 mcg/ml(HRV3-1), 5 mcg/ml (HRV3-2), and 0.5 mcg/ml (HRV3-3) on the integrity oftissue infected with Rhinovirus A16. TEER was monitored 24 (D1) and 48hours (D2) post-inoculation on MucilAir™ 3D media.

FIG. 4 illustrates the effect of treatment with a combination ofapolactoferrin, lysozyme, and soluble ICAM-1 at the three differentdoses shown in Table 5 (HRV4-1, HRV4-2, and HRV4-3), on the integrity oftissue infected with Rhinovirus A16. TEER was monitored 24 (D1) and 48hours (D2) post-inoculation on MucilAir™ 3D media.

FIG. 5 illustrates the effect of apolactoferrin treatment at 500 mcg/ml(HRV1-1), 50 mcg/ml (HRV1-2), and 5 mcg/ml (HRV1-3) on LDH release ofcells infected with Rhinovirus A16. Cytotoxicity was monitored 24 (D1)and 48 (D2) hours post-inoculation on MucilAir™ 3D media.

FIG. 6 illustrates the effect of lysozyme treatment at 2500 mcg/ml(HRV2-1), 250 mcg/ml (HRV2-2), and 25 mcg/ml (HRV2-3) on LDH release ofcells infected with Rhinovirus A16. Cytotoxicity was monitored 24 (D1)and 48 (D2) hours post-inoculation on MucilAir™ 3D media.

FIG. 7 illustrates the effect of soluble ICAM-1 treatment at 50 mcg/ml(HRV3-1), 5 mcg/ml (HRV3-2), and 0.5 mcg/ml (HRV3-3) on LDH release ofcells infected with Rhinovirus A16. Cytotoxicity was monitored 24 (D1)and 48 (D2) hours post-inoculation on MucilAir™ 3D media.

FIG. 8 illustrates the effect of a combination of apolactoferrin,lysozyme, and soluble ICAM-1 at the three different doses shown in Table5 (HRV4-1, HRV4-2, and HRV4-3) on LDH release infected with RhinovirusA16. Cytotoxicity was monitored 24 (D1) and 48 (D2) hourspost-inoculation on MucilAir™ 3D media.

FIG. 9 illustrates the effect of apolactoferrin treatment at 500 mcg/ml(HRV1-1), 50 mcg/ml (HRV1-2), and 5 mcg/ml (HRV1-3) on cilia beating.Cilia beating frequency was monitored 24 (D1) and 48 (D2) hourspost-inoculation on MucilAir™ 3D media.

FIG. 10 illustrates the effect of Rhinovirus A16 infection on ciliabeating frequency of epithelial cells with lysozyme treatment at 2500mcg/ml (HRV2-1), 250 mcg/ml (HRV2-2), and 25 mcg/ml (HRV2-3). Ciliabeating frequency was monitored 24 (D1) and 48 (D2) hourspost-inoculation on MucilAir™ 3D media.

FIG. 11 illustrates the effect of Rhinovirus A16 infectionon ciliabeating frequency of epithelial cells with soluble ICAM-1 treatment at50 mcg/ml (HRV3-1), 5 mcg/ml (HRV3-2), and 0.5 meg/ml (HRV3-3). Ciliabeating frequency was monitored 24 (D1) and 48 (D2) hourspost-inoculation on MucilAir™ 3D media.

FIG. 12 illustrates the effect of Rhinovirus A16 infection on ciliabeating frequency of epithelial cells with a combination ofapolactoferrin, lysozyme, and soluble ICAM-1 at the three differentdoses shown in Table 5 (HRV4-1, HRV4-2, and HRV4-3). Cilia beatingfrequency was monitored 24 (D1) and 48 (D2) hours post-inoculation onMucilAir™ 3D media.

FIG. 13 illustrates the effect of Rhinovirus A16 infection onmucociliary clearance of epithelial cells with HRV treatments.Mucociliary clearance was monitored 48 (D2) hours post-inoculation onMucilAir™ 3D media.

FIG. 14 illustrates the genome copy number of Rhinovirus A16 infectionwith apolactoferrin treatment at 500 mcg/ml (HRV1-1), 50 mcg/ml(HRV1-2), and 5 mcg/ml (HRV1-3). Viral load was measured at 3.5, 24, and48 hours post-inoculation on MucilAir™ 3D media.

FIG. 15 illustrates the genome copy number of Rhinovirus A16 infectionwith lysozyme treatment at 2500 mcg/ml (HRV2-1), 250 mcg/ml (HRV2-2),and 25 mcg/ml (HRV2-3). Viral load was measured at 3.5, 24, and 48 hourspost-inoculation on MucilAir™ 3D media.

FIG. 16 illustrates the genome copy number of Rhinovirus A16 infectionwith soluble ICAM-1 treatment at 50 mcg/ml (HRV3-1), 5 mcg/ml (HRV3-2),and 0.5 mcg/ml (HRV3-3). Viral load was measured at 3.5, 24, and 48hours post-inoculation on MucilAir™ 3D media.

FIG. 17 illustrates the genome copy number of Rhinovirus A16 infectionwith a combination of apolactoferrin, lysozyme, and soluble ICAM-1 atthe three different doses shown in Table 5 (HRV4-1, HRV4-2, and HRV4-3).Viral load was measured at 3.5, 24, and 48 hours post-inoculation onMucilAir™ 3D media.

FIG. 18 illustrates the mucin quantity as measured with an ELLA assayfrom the apical medium with apolactoferrin treatment at 500 mcg/ml(HRV1-1), 50 mcg/ml (HRV1-2), and 5 mcg/ml (HRV1-3) and Rhinovirus A16infection at 24 (D1) and 48 hours (D2) post-inoculation on MucilAir™ 3Dmedia.

FIG. 19 illustrates the mucin quantity as measured with an ELLA assayfrom the apical medium with lysozyme treatment at 2500 mcg/ml (IAV2-1),250 mcg/ml (HRV2-2), and 25 meg/ml (HRV2-3) and Rhinovirus A16 infectionat 24 (D1) and 48 hours (D2) post-inoculation on MucilAir™ 3D media.

FIG. 20 illustrates the mucin quantity as measured with an ELLA assayfrom the apical medium with soluble ICAM-1 treatment at 50 mcg/ml(HRV3-1), 5 mcg/ml (HRV3-2), and 0.5 mcg/ml (HRV3-3) and Rhinovirus A16infection at 24 (D1) and 48 hours (D2) post-inoculation on MucilAir™ 3Dmedia.

FIG. 21 illustrates the mucin quantity as measured with an ELLA assayfrom the apical medium with apolactoferrin treatment at 500 mcg/ml(HRV1-1), 50 mcg/ml (HRV1-2), and 5 mcg/ml (HRV1-3) and Rhinovirus A16infection at 24 (D1) and 48 hours (D2) post-inoculation on MucilAir™ 3Dmedia.

FIG. 22 illustrates the effect of Influenza A H1N1 infection on tissueintegrity with apolactoferrin treatment at 500 mcg/ml (IAV1-1), 50mcg/ml (IAV1-2), and 5 mcg/ml (IAV1-3). TEER was monitored 24 (D1) and48 hours (D2) post-inoculation on MucilAir™ 3D media.

FIG. 23 illustrates the effect of Influenza A H1N1 infection on tissueintegrity with lysozyme treatment at 2500 mcg/ml (IAV2-1), 250 mcg/ml(IAV2-2), and 25 mcg/ml (IAV2-3). TEER was monitored 24 (D1) and 48 (D2)hours post-inoculation on MucilAir™ 3D media.

FIG. 24 illustrates the effect of Influenza A H1N1 infection on tissueintegrity with a combination of 3′-sialyllactose and 6′sialyllactosetreatment each at 327 mcg/ml (IAV3-1), 3.27 mcg/ml (IAV3-2), and 0.327mcg/ml (IAV3-3). TEER was monitored 24 (D1) and 48 (D2) hourspost-inoculation on MucilAir™ 3D media.

FIG. 25 illustrates the effect of Influenza A H1N1 infection on tissueintegrity with a combination of apolactoferrin, lysozyme, andsialyllactoses at the three different doses shown in Table 5 (IAV4-1,IAV4-2, and IAV4-3). TEER was monitored 24 (D1) and 48 (D2) hourspost-inoculation on MucilAir™ 3D media.

FIG. 26 illustrates the effect of Influenza A H1N1 infection on LDHrelease from epithelial cells apolactoferrin treatment at 500 mcg/ml(IAV1-1), 50 mcg/ml (IAV1-2), and 5 mcg/ml (IAV1-3). Cytotoxicity wasmonitored 24 (D1) and 48 (D2) hours post-inoculation on MucilAir™ 3Dmedia.

FIG. 27 illustrates the effect of Influenza A H1N1 infection on LDHrelease from epithelial cells with lysozyme treatment at 2500 mcg/ml(IAV2-1), 250 mcg/ml (IAV2-2), and 25 mcg/ml (IAV2-3). Cytotoxicity wasmonitored 24 (D1) and 48 (D2) hours post-inoculation on MucilAir™ 3Dmedia.

FIG. 28 illustrates the effect of Influenza A H1N1 infection on LDHrelease from epithelial cells with a combination of 3′-sialyllactose and6′sialyllactose treatment each at 327 mcg/ml (IAV3-1), 3.27 mcg/ml(IAV3-2), and 0.327 mcg/ml (IAV3-3). Cytotoxicity was monitored 24 (D1)and 48 (D2) hours post-inoculation on MucilAir™ 3D media.

FIG. 29 illustrates the effect of Influenza A H1N1 infection on LDHrelease from epithelial cells with a combination of apolactoferrin,lysozyme, and sialyllactoses at the three different doses shown in Table5 (IAV4-1, IAV4-2, and IAV4-3). Cytotoxicity was monitored 24 (D1) and48 (D2) hours post-inoculation on MucilAir™ 3D media.

FIG. 30 illustrates the effect of Influenza A H1N1 infection on ciliabeating frequency of epithelial cells with apolactoferrin treatment at500 mcg/ml (IAV1-1), 50 mcg/ml (IAV1-2), and 5 mcg/ml (IAV1-3). Ciliabeating frequency was monitored 24 (D1) and 48 (D2) hourspost-inoculation on MucilAir™ 3D media.

FIG. 31 illustrates the effect of Influenza A H1N1 infection on ciliabeating frequency of epithelial cells with lysozyme treatment at 2500mcg/ml (IAV2-1), 250 mcg/ml (IAV2-2), and 25 mcg/ml (IAV2-3). Ciliabeating frequency was monitored 24 (D1) and 48 (D2) hourspost-inoculation on MucilAir™ 3D media.

FIG. 32 illustrates the effect of Influenza A H1N1 infectionon ciliabeating frequency of epithelial cells with a combination of3′-sialyllactose and 6′sialyllactose treatment each at 327 mcg/ml(IAV3-1), 3.27 mcg/ml (IAV3-2), and 0.327 mcg/ml (IAV3-3). Cilia beatingfrequency was monitored 24 (D1) and 48 (D2) hours post-inoculation onMucilAir™ 3D media.

FIG. 33 illustrates the effect of Influenza A H1N1 infection on ciliabeating frequency of epithelial cells with a combination ofapolactoferrin, lysozyme, and sialyllactoses at the three differentdoses shown in Table 5 (IAV4-1, IAV4-2, and IAV4-3). Cilia beatingfrequency was monitored 24 (D1) and 48 (D2) hours post-inoculation onMucilAir™ 3D media.

FIG. 34 illustrates the effect of Influenza A H1N1 infection onmucociliary clearance of epithelial cells with IAV treatments.Mucociliary clearance was monitored 48 (D2) hours post-inoculation onMucilAir™ 3D media.

FIG. 35 illustrates the genome copy number of Influenza A H1N1 infectionwith apolactoferrin treatment at 500 mcg/ml (IAV1-1), 50 mcg/ml(IAV1-2), and 5 mcg/ml (IAV1-3). Viral load was measured at 3.5, 24, and48 hours post-inoculation on MucilAir™ 3D media.

FIG. 36 illustrates the genome copy number of Influenza A H1N1 infectionwith lysozyme treatment at 2500 mcg/ml (IAV2-1), 250 mcg/ml (IAV2-2),and 25 mcg/ml (IAV2-3). Viral load was measured at 3.5, 24, and 48 hourspost-inoculation on MucilAir™ 3D media.

FIG. 37 illustrates the genome copy number of Influenza A H1N1 infectionwith a combination of 3′-sialyllactose and 6′sialyllactose treatmenteach at 327 mcg/ml (IAV3-1), 3.27 mcg/ml (IAV3-2), and 0.327 mcg/ml(IAV3-3). Viral load was measured at 3.5, 24, and 48 hourspost-inoculation on MucilAir™ 3D media.

FIG. 38 illustrates the genome copy number of Influenza A H1N1 infectionwith a combination of apolactoferrin, lysozyme, and sialyllactoses atthe three different doses shown in Table 5 (IAV4-1, IAV4-2, and IAV4-3).Viral load was measured at 3.5, 24, and 48 hours post-inoculation onMucilAir™ 3D media.

FIG. 39 illustrates the mucin quantity as measured with an ELLA assayfrom the apical medium with apolactoferrin treatment at 500 mcg/ml(IAV1-1), 50 mcg/ml (IAV1-2), and 5 mcg/ml (IAV1-3) and Influenza A H1N1infection at 24 (D1) and 48 (D2) hours post-inoculation on MucilAir™ 3Dmedia.

FIG. 40 illustrates the mucin quantity as measured with an ELLA assayfrom the apical medium with lysozyme treatment at 2500 mcg/ml (IAV2-1),250 mcg/ml (IAV2-2), and 25 mcg/ml (IAV2-3) and Influenza A H1N1infection at 24 (D1) and 48 (D2) hours post-inoculation on MucilAir™ 3Dmedia.

FIG. 41 illustrates the mucin quantity as measured with an ELLA assayfrom the apical medium with a combination of 3′-sialyllactose and6′sialyllactose treatment each at 327 mcg/ml (IAV3-1), 3.27 mcg/ml(IAV3-2), and 0.327 mcg/ml (IAV3-3) and Influenza A H1N1 infection at 24(D1) and 48 (D2) hours post-inoculation on MucilAir™ 3D media.

FIG. 42 illustrates the mucin quantity as measured with an ELLA assayfrom the apical medium with a combination of apolactoferrin, lysozyme,and sialyllactoses at the three different doses shown in Table 5(IAV4-1, IAV4-2, and IAV4-3) and Influenza A H1N1 infection at 24 (D1)and 48 (D2) hours post-inoculation on MucilAir™ 3D media.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure may be understood more readily by reference tothe following detailed description and the Examples included therein.Before the present methods and techniques are disclosed and described,it is to be understood by one of skill in the art that this disclosureis not to be limited to the specific analytical or synthetic methodsdescribed herein. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting. Unless defined otherwise, all technicaland scientific terms used herein have the meaning commonly understood byone of ordinary skill in the art to which this disclosure belongs.

By “agent” or “therapeutic agent” is meant any small molecule chemicalcompound, antibody, nucleic acid molecule, or polypeptide or fragmentsthereof. By “therapeutic agent” is meant any of the compositionsdedicated to preventing or treating respiratory infections describedherein.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a respiratorydisease or a symptom thereof.

By “analog” is meant a molecule that is not identical, but has analogousfunctional or structural features. For example, a polypeptide analogretains the biological activity of a corresponding naturally-occurringpolypeptide, while having certain biochemical modifications that enhancethe analog's function relative to a naturally occurring polypeptide.Such biochemical modifications could increase the analog's proteaseresistance, membrane permeability, or half-life, without altering, forexample, ligand binding. An analog may include an unnatural amino acid.

As used herein the term “prophylaxis” refers to preventing, diminishingthe extent of or retarding the rate of infection (e.g. viral infection,etc.).

As used herein “an interfering RNA” refers to any double stranded orsingle stranded RNA sequence, capable-either directly or indirectly(i.e., upon conversion)—of inhibiting or down regulating gene expressionby mediating RNA interference. Interfering RNA includes, but is notlimited to, small interfering RNA (“siRNA”) and small hairpin RNA(“shRNA”). “RNA interference” refers to the selective degradation of asequence-compatible messenger RNA transcript.

As used herein “an shRNA” (small hairpin RNA) refers to an RNA moleculecomprising an antisense region, a loop portion and a sense region,wherein the sense region has complementary nucleotides that base pairwith the antisense region to form a duplex stem. Followingpost-transcriptional processing, the small hairpin RNA is converted intoa small interfering RNA by a cleavage event mediated by the enzymeDicer, which is a member of the RNase III family.

As used herein “an RNAi” (RNA interference) refers to apost-transcriptional silencing mechanism initiated by smalldouble-stranded RNA molecules that suppress expression of genes withsequence homology.

As used herein, “changed as compared to a control” sample or subject isunderstood as having a level of the analytic or diagnostic ortherapeutic indicator to be detected at a level that is statisticallydifferent than a sample from a normal, untreated, or control sample orsubject. Control samples include, for example, cells in culture, one ormore laboratory test animals, or one or more human subjects. Methods toselect and test control samples are within the ability of those skilledin the art. An analytic substance can be a naturally occurring substancethat is characteristically expressed or produced by the cell or organism(e.g., antibodies, pathogenic peptides or particles, and the like) or asubstance produced by a reporter construct (e.g, β-galactosidase orluciferase). Depending on the detection method used, the amount andmeasurement of the change may vary. Determination of statisticalsignificance is within the ability of those skilled in the art.

As used herein, the term “co-administering,” or “co-administration,” andthe like refers to the act of administering two or more agents (e.g., anantimicrobial agent and an anti-viral agent), compounds, therapies, orthe like, at or about the same time. The order or sequence ofadministering the different agents of the disclosure, e.g., antibiotics,antivirals, antifungals, or immunotherapeutic agents, may vary and isnot confined to any particular sequence. Co-administering may also referto the situation where two or more agents are administered to differentregions of the body or via different delivery schemes, e.g., where afirst agent is administered intranasally and a second agent isadministered systemically, or vice versa. Co-administering may alsorefer to two or more agents administered via the same delivery scheme,e.g., where a first agent is administered intranasally and a secondagent is administered intranasally.

As used herein, the terms “comprises,” “comprising,” “containing” and“having” and the like are open-ended as defined by U.S. Patent law andcan mean “includes,” “including,” and the like. The terms “consistingessentially of” or “consists essentially” likewise have the meaningascribed to them in U.S. Patent law and are open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited are not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

“Contacting a cell” is understood herein as providing an agent to a cell(e.g. a nasal membrane cell), such that the agent may interact with thecell (e.g., nasal membrane cell to be treated) and/or taken up by thecell, and have an effect on the cell. The agent (e.g., an antimicrobialor antiviral agent) may be delivered to the cell directly (e.g., byaddition of the agent to a gel or aerosol formulation for nasaldelivery. One of ordinary skill in the art will readily understand thatadministration of a therapeutic agent to a subject involves contactingthe therapeutic agent with a cell or tissue of the subject.

As used herein, the term “coupled,” as in reference to two or moreagents being “coupled” together, refers to a covalent or otherwisestable association between the two or more agents. For example, atherapeutic agent may be coupled with an antimicrobial agent via acovalent bond, a covalently tethered linker moiety, or non-covalentlythrough ionic interactions or hydrogen bonding. One or more agents thatare coupled together retain substantially their same independentfunctions and characteristics. For example, the therapeutic agent whencoupled to another agent may retain its same activity as if it wereindependent.

By “cycle” or “drug cycle” is meant the administration of repetitivedosing for a defined period of time, which may range from minutes tohours to days to weeks to months or even years.

By “cytokine” is meant a hormone that acts locally and that modulates anindividual's to immune response.

As used herein, “detecting,” “detection” and the like are understood toinclude an assay performed to determine one or more characteristics of asample, e.g. identifying the presence, absence or amount of the analyteto be detected. For example, detection may include identification of aspecific analyte in a sample or an activity of an agent in a sample.Detection may include the determination of the presence of nucleic acidor protein (e.g., antibody, cytokine, and the like) by PCR, immunoassay(e.g., ELISA, ELLA, etc.), microscopy, pathogen challenge, and the like.The amount of analyte or activity detected in the sample may be none orbelow the level of detection of the assay or method.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ. Anexemplary disease is a respiratory infection.

The terms “effective amount,” “therapeutically effective amount” or“pharmaceutically effective amount” as used herein, refer to an amountof an agent or compound that is sufficient to prevent or treat adisorder, e.g., a cancer. In some embodiments, the result is a reductionin and/or alleviation of the signs, symptoms, or causes of a disorder,or any other desired alteration of a biological system. For example, an“effective amount” for therapeutic may be the amount of the compositioncomprising a compound as disclosed herein required to provide aclinically significant decrease in a disease/disorder (e.g. arespiratory infection). An “effective amount” or therapeuticallyeffective amount of an agent or combination of agents of the disclosuremay also be that amount or dose that is effective to substantiallyshrink or eliminate an infection, or prevent its occurrence. Anappropriate “effective” amount in any individual case is determinedusing any suitable technique, (e.g., a dose escalation study) and willdepend on the judgment of the practitioner. However, suitable dosageranges are readily determinable by to one skilled in the art.

More than one dose may be required to provide an effective dose. It isunderstood that an effective dose in one population may or may not besufficient in all populations. Thus, in connection with theadministration of a therapeutic agent, the therapeutic agent may be“effective against” a disease or condition when administration in aclinically appropriate manner results in a beneficial effect for atleast a statistically significant fraction of subjects, such as aprevention of disease onset, improvement of symptoms, a cure, areduction in disease signs or symptoms, extension of life, improvementin quality of life, or other effect generally recognized as positive bymedical doctors familiar with treating the particular type of disease orcondition.

By “enhances” is meant a positive alteration of at least 10%, 25%, 50%,75%, 100%, or any number there between.

As used herein, an “immunoassay” is a detection method based on thespecific binding of at least one antibody to an antigen, e.g., ELISA,ELLA, RIA, western blot, and the like.

As used herein “immunogen,” “immunogenic,” and the like refer tosubstances that can promote an immune response, e.g., an antibody basedor cell mediated immune response, in at least one organism.

By “immunogenic composition” is meant a composition comprising amolecule capable of inducing or modulating an immune response in asubject. Such an immune response may be a prophylactic or therapeuticimmune response.

As used herein, the term “immunotherapeutic agent” refers to any agent,compound, or biologic that is capable of modulating the host's immunesystem. For example, an immunotherapeutic agent is capable of causing astimulation of the immune system against a respiratory infection.

As used herein “inducing immunity” is meant to refer to any immuneresponse generated against an antigen. In embodiments, immunity ismediated by antibodies against an infectious agent, which is exhibitedby a vertebrate (e.g., a human), that prevents or ameliorates aninfection or reduces at least one symptom thereof. The immunogeniccompositions of the disclosure can stimulate the production ofantibodies that, for example, neutralize airborne pathogens/infectiousagents, block infectious agents from entering cells, block replicationof infectious agents, and/or protect host cells from infection anddestruction. The term can also refer to an immune response that ismediated by T-lymphocytes and/or other white blood cells against aninfectious agent, exhibited by a vertebrate (e.g., a human), thatprevents or ameliorates an infection or reduces at least one symptomthereof.

The term “isolated”, as used herein, refers to any composition,molecule, or mixture that has undergone a laboratory purificationprocedure including, but not limited to, extraction, centrifugation,chromatographic separation (i.e., for example, thin layer chromatographyor high performance liquid chromatography). Usually such a purificationprocedure provides an isolated composition, molecule, or mixture basedupon physical, chemical, or electrical potential properties. Dependingupon the choice of procedure an isolated composition, molecule, ormixture may contain other compositions, compounds or mixtures havingsimilar chemical properties. For example, an isolated composition,molecule, or mixture may contain between 1-20%, 1-10%, or 1-5% ofcompositions or mixtures having similar chemical properties.

As used herein, the term “local” or “locally,” as in localadministration or coadministration of one or more therapeutics, refersto the delivery of a therapeutic agent to a bodily site (e.g. a nasalmembrane) that is proximate or nearby the site of an infection, adjacentor immediately nearby the site of the infection, at the perimeter of orin contact with the infection, or within or inside the infected tissue.Local administration generally excludes systemic administration routes.

As used herein, “nucleic acid” as in a nucleic acid for delivery to acell is understood by its usual meaning in the art as a polynucleotideor oligonucleotide that refers to a string of at least twobase-sugar-phosphate combinations. Nucleotides are the monomeric unitsof nucleic acid polymers. The term includes deoxyribonucleic acid (DNA)and ribonucleic acid (RNA) in the form of an oligonucleotide messengerRNA, anti-sense, plasmid DNA, parts of a plasmid DNA, genetic materialderived from a virus, and the like. Polynucleotides include nucleicacids of at least two monomers. Anti-sense polynucleotides are nucleicacids that interfere with the function of DNA or RNA. An siRNA or anshRNA is a double stranded RNA that inhibits or disrupts activity ortranslation, for example by promoting degradation of modifying splicingor processing of the cellular nucleic acid, e.g., mRNA, microRNA, andthe like, to which it is targeted. As used herein, siRNA and shRNAinclude any double stranded RNA molecule that can modulate thestability, translation, or splicing of an RNA to which at least onestrand of the double stranded nucleic acid hybridizes. RNAs are wellknown in the art, see e.g., patent publications WO/2002/044321,WO/2003/099298, US 20050277610, US 20050244858; and U.S. Pat. Nos.7,297,786, 7,560,438 and 7,056,704, all of which are incorporated hereinby reference. Nucleic acid as used herein is understood to includenon-natural nucleotides (not occurring in nature), for example: aderivative of natural nucleotides such as phosphothionates or peptidenucleic acids (such as those described in the patents and applicationscited immediately above). A nucleic acid can be delivered to a cell inorder to produce a cellular change that is therapeutic or prophylactic.The nucleic acid may express a protein or polypeptide, e.g., a proteinthat is missing or non-functional in the cell or subject. The nucleicacid may be single or double stranded, may be sense or anti-sense, andcan be delivered to a cell as naked DNA, in combination with agents topromote nucleic acid uptake to into a cell (e.g., transfectionreagents), in the context of a viral vector, and the like. The nucleicacid can be targeted to a nucleic acid that is endogenous to the cell(mRNA or microRNA), or a heterologous nucleic acid (e.g., nucleic acidfrom a pathogen, such as a viral gene). Delivery of a nucleic acid meansto transfer a nucleic acid from outside a subject to within the outercell membrane of a cell in the subject.

“Obtaining” is understood herein as manufacturing, purchasing,synthesizing, isolating, purifying, or otherwise coming into possessionof.

The term “pharmaceutically acceptable” as used herein, refers to amaterial, (e.g., a carrier or diluent), which does not abrogate thebiological activity or properties of the compounds described herein, andis relatively nontoxic (i.e., the material is administered to anindividual without causing undesirable biological effects or interactingin a deleterious manner with any of the components of the composition inwhich it is contained).

The phrase “pharmaceutically acceptable carrier, excipient, or diluent”is art recognized and includes a pharmaceutically acceptable material,composition or vehicle, suitable for administering compounds of thepresent disclosure to mammals. As used herein, the term“pharmaceutically acceptable” means being approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopia, European Pharmacopia or other generally recognizedpharmacopia for use in mammals, e.g., humans.

As used herein, the term “pharmaceutically effective regimen” refers toa systematic plan for the administration of one or more therapeuticagents, which includes aspects such as type of therapeutic agent,therapeutic agent concentrations, and any changes therein made duringthe course of the drug administration, which when administered iseffective in treating and/or preventing an infection. Suchconsiderations depend on the judgment of the practitioner and arereadily determinable by one skilled in the art.

A “polypeptide” or “peptide” as used herein is understood as two or moreindependently selected natural or non-natural amino acids joined by acovalent bond (e.g., a peptide bond). A peptide can include 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more naturalor non-natural amino acids joined by peptide bonds. Polypeptides asdescribed herein include full length proteins (e.g., fully processedproteins) as well as shorter amino acids sequences (e.g., fragments ofnaturally occurring proteins or synthetic polypeptide fragments).

Ranges provided herein are understood to be shorthand for all of thevalues within the range including the limits of the range. For example,a range of 1 to 50 is understood to include any number, combination ofnumbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal valuesbetween the aforementioned integers such as, for example, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nestedsub-ranges” that extend from either end point of the range arespecifically contemplated. For example, a nested sub-range of anexemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 inthe other direction.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%,75%, 100%, or any number there between.

By “reference” is meant a standard or control condition.

As used herein, the term “regimen” refers to the various parameters thatcharacterize how a drug or agent is administered, including, the dosagelevel, timing, and iterations, as well as the ratio of different drugsor agents to one another. The term “pharmaceutically effective regimen”refers to a particular regimen that provides a desired therapeuticresult or effect. The term “iterations” refer to the general concept ofrepeating sets of administering one or more agents. For example, acombination of drug X and drug Y may be given (co-administered at orabout at the same time and in any order) to a patient on a first day atdose Z. Drugs X and Y may then be administered (co-administered at orabout at the same time and in any order) again at dose Z, or anotherdose, on a second day. The timing between the first and second days canbe 1 day or anywhere up to several days, or a week, or several weeks, ormonths. The iterative administrations may also occur on the same day,separated by a specified number of minutes (e.g., 10 minutes, 20minutes, 30 minutes or more) or hours (e.g., 1 hour, 2 hours, 4 hours, 6hours, 12 hours). An effective dosing regimen may be determinable bythose of ordinary skill in the art, e.g., prescribing physician, usingstandard practices.

A “sample” as used herein refers to a biological material that isisolated from its environment (e.g., blood or tissue from an animal,cells, or conditioned media from tissue culture). In embodiments, thesample is suspected of containing, or known to contain an analyte, suchas an infectious agent or a protein of interest (e.g., antibody,cytokine, and the like). A sample can also be a partially purifiedfraction of a tissue or bodily fluid. A reference sample can be a“normal” sample, from a donor not having the disease or condition fluid,or from a normal tissue in a subject having the disease or condition, oran untreated to subject (e.g., a subject not treated with the vaccine).A reference sample can also be taken at a “zero time point” prior tocontacting the cell or subject with the agent or therapeuticintervention to be tested.

As used herein, the term “selectively” means tending to occur at ahigher frequency in one population than in another population.

By “specifically binds” is meant recognition and binding to a target(e.g., polypeptide, cell, and the like), while not substantiallyrecognizing and/or binding other molecules in a sample, for example, abiological sample.

The term “subject”, as used herein, refers to any organism that iscapable of experiencing a respiratory infection. Such organisms include,but are not limited to, human, dog, cat, horse, cow, sheep, goat, mouse,rat, guinea pig, monkey, primate, non-human primate, avian, reptilesetc.

A subject “suffering from or suspected of suffering from” a specificdisease, condition, or syndrome (e.g., a respiratory infection) has asufficient number of risk factors or presents with a sufficient numberor combination of signs or symptoms of the disease, condition, orsyndrome such that a competent individual would diagnose or suspect thatthe subject was suffering from the disease, condition, or syndrome.Methods for identification of subjects suffering from or suspected ofsuffering from respiratory infection is within the ability of those inthe art. Subjects suffering from, and suspected of suffering from, aspecific disease, condition, or syndrome are not necessarily twodistinct groups.

As used herein, “susceptible to” or “prone to” or “predisposed to” aspecific disease or condition and the like refers to an individual whobased on genetic, environmental, health, and/or other risk factors ismore likely to develop a disease or condition than the generalpopulation. An increase in likelihood of developing a disease may be anincrease of about 10%, 20%, 50%, 100%, 150%, 200%, or more.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease.

The term “glycolipids”, as used herein, refers to any molecule with atleast one carbohydrate chain linked to a ceramide, a fatty acid chain,or any other lipid. Alternatively, a glycolipid maybe referred to as aglycosphingolipid.

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural reference unless the context clearlydictates otherwise. Thus, for example, reference to “a gene” is areference to one or more genes and includes equivalents thereof known tothose skilled in the art, and so forth.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein can be modified by theterm about.

Unless otherwise indicated, all references to concentrations include theindicated amounts on a weight by weight, weight by volume or volume byvolume basis. Any reference to a percent concentration will beunderstood to refer to wt/wt, wt/vol, or wt/vol. While certainembodiments may be described by concentrations as wt/wt or wt/vol, itshould be understood that such compositions disclose the same % on awt/wt or wt/vol basis. The density of any forms of the invention may bebetween 0.8 g/mL and 1.2 g/mL, for example between 0.9 g/mL and 1.1 g/mLor between 0.95 g/mL and 1.05 g/mL.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Other definitions appear in context throughout this disclosure.

Any therapeutic agents, compositions, or methods provided herein can becombined with one or more of any of the other therapeutic agents,compositions, and methods provided herein.

The present disclosure provides compositions and methods for preventingand treating respiratory infections. The present disclosure featuresmethods and compositions for enhancing the filtering capabilities of thenasal membranes and protecting against airborne pathogens by enhancingthe health of the nasal membranes and filtering capabilities of nasalmucous. In particular, the disclosure features antimicrobial, antiviral,and antifungal compositions that prevent and treat respiratoryinfections caused by bacteria, viruses, and fungi, including influenzaviruses and rhinoviruses (e.g. viruses that cause the flu and commoncold, respectively). The present disclosure is based, at least in part,on the discovery that compositions including antimicrobial, antiviral,and/or antifungal functionalities may be used to enhance the health andfiltering capabilities of nasal membranes and protect against airbornepathogens. In so doing, they must also maintain the physiological healthof the membranes such as by maintaining a healthy Ph and osmolarity andencouraging the propagation of healthy microflora. In a particularexemplary embodiment, the present disclosure relates to an antimicrobialand anti-fungal filtering composition formulated for topical applicationto the proximal anterior nares or the inner anterior nasal membrane,where it may also coat nasal hairs and enhance the filteringcapabilities of the nose. Advantageously, the present disclosure, asdescribed herein, provides a topically applied filtering composition fornasal and/or oral application that does not adversely affect thechemical properties of the respiratory membranes or mucosa (and enhancesits natural filtering capabilities) and that will specifically targetand protect against several disease causing microorganisms.

The present disclosure also provides for methods of enhancing thenatural filtration properties of the respiratory membranes and reducingthe number of microorganisms, allergens, and odorants entering the bodythrough the nose or proliferating along the respiratory membranes. Insome embodiments, this method includes application of a topical orinhaled, or ingested solution of antimicrobial, antiviral, anti-fungal,and/or odor-neutralizing composition to the mouth, the throat, theopening of the nostrils, the nasal epithelial inside the nostrils,and/or the nasal hairs. The antimicrobial, antiviral, and anti-fungalsolution may be in gel, lotion, lozenge, vapor, or aerosol forms and mayhave a combination of active ingredients intended to bolster the naturalfiltration capabilities of the nose in a base medium that allows theactive ingredients to be well tolerated, in their active forms, whilepreventing them from having undesirable effects. The compositions hereinmay also mimic the chemical properties of natural healthy mucous orsaliva such as, for example, pH and osmolarity. The ingredients may bebalanced to create a synergistic effect stronger than any of theingredients alone and may also be balanced to maintain a healthy pH andosmolarity in the respiratory membranes as these parameters have beenshown to affect the likelihood of disease transmission and allergicreaction. The active ingredients may include, but are not limited to,recombinant, naturally derived, or purified lactoferrin, lysozyme, ICAM,cationic peptides, glycosylated peptides, sialic acid, quercetin, orother bioflavonoids, in addition to any plant extract havingantimicrobial properties (including but not limited to fruit peelextracts and carrageenans), silver, copper, or zinc microparticles, andlauric acid, which have proven antimicrobial properties. Variousembodiments may also include one or more of the following ingredients:sodium bicarbonate, activated charcoal, cocoa butter, shea butter,beeswax, plant butters, glycerine, honey, alginates, or plant mucilage,and a preservative such as vitamin C, vitamin E, or rosmarinic acid.Various embodiments may include ingredients that, when mixed with theaforementioned active ingredients, create a formulation that iswell-tolerated when applied to the opening of the nostrils, the nasalepithelia inside the nostrils, or the nasal hairs, and that allows theactive ingredients to adhere to the areas of application for asufficient period of time to prevent infection or allergy beforereapplication.

Respiratory Function

Respiratory infections are among the most common type of communicablediseases throughout the world today. Almost yearly, new and potentiallydeadly diseases such as Middle East Respiratory Syndrome (MERS) andAvian and Swine influenzas capture world-wide attention and concern. Newand unusual strains of influenza virus are continuously emerging and arecapable of creating worldwide epidemics in a matter of months. Moreover,the current state of vaccine and anti-viral technology is not wellequipped to deal with these outbreaks in a timely manner. At best, atargeted vaccine to a new viral strain is available in six months to ayear, at which point an epidemic could be well underway.

Every day, about 12,000 liters of air is filtered by the average nose.The nasal passages filter 95% of particles greater than 15 μm indiameter out of the air. The are normally trapped by mucous and theningested. Micro-organisms and allergens are normally several orders ofmagnitude smaller than this threshold and have evolved to evade orovercome natural mucosal defenses in the nose, penetrate the nasalmembranes, and/or enter the lower respiratory tract via the mouth orthroat. According to the techniques herein, augmenting the respiratorymembranes and mucosa with anti-microbial and filtering agents may allowa significant number of respiratory infections and allergies to beprevented in an easy, unobtrusive, and convenient way. Additionally,many of the undesirable particles entering the nose are odorants whichcan be a nuisance and which can be filtered or neutralized by certainsubstances such as but not limited to activated charcoal or sodiumbicarbonate suspended in a carrier before they are able to bind odorantreceptors.

The present disclosure is directed to an antimicrobial, antiviral,anti-fungal, odor-neutralizing topical application that simulatescertain chemical properties of nasal mucous, does not impair the healthor integrity of the nasal membranes, or adversely affect its beneficialmicroflora, and also serves as a filter to prevent airborne irritantsand pathogens from penetrating the nasal membranes and/or entering thelower respiratory tract. In so doing, the compositions herein preventinfection of the respiratory tract, while also preventing irritationand/or allergic reactions.

In addition, the compositions herein may be isotonic to nasal epitheliaand mucous membranes and contain compounds with health promotingproperties. Beneficial microflora and certain properties of the nasalmembrane such as osmolarity and pH have been shown to affect thelikelihood of infection. Several medications and health conditions havebeen identified that make people more susceptible to respiratoryinfection. For example, diabetics are likely to have dry nasal membranesand suffer from fungal sinusitis. Oral contraceptives, sleep apneamachines, and allergies are also known to make the nasal membranes drierand more susceptible to infection.

The compositions of the present disclosure may include specific activeingredients with proven antimicrobial properties including but notlimited to, ICAM-1, ICAM-1 inhibitors, sialic acid, neuraminidaseinhibitors, lysozyme, lactoferrin, citrus oils, extracts, orderivatives, plant mucilage, peptides, glycopeptides, amino acids,antimicrobial oils, antimicrobial plant extracts, or defensins. Thecomposition may include odor-neutralizing compounds such as, but notlimited to activated charcoal or sodium bicarbonate. The composition mayhave adhesive properties and be specifically formulated to keep activeingredients and antimicrobials/antivirals on the surface of the nasalepithelium for an extended period of time. To achieve this, thecompositions herein may include substances of low volatility, orocclusive substances such as, for example, polyols, shea butter, orother plant butters, coconut oil, beeswax, and bioadhesive substancessuch as mucilage, or alginates.

The nasal formulations described herein and their active ingredients areintended to be well tolerated, exert beneficial effect on ciliaryfunction, have good dispensing properties, a high degree of adhesion,and maintain the chemical properties of the mucosa. In some embodiments,ingredients are balanced to create synergistic effects.

According to the techniques herein, one or more of the activeingredients may target undesirable microorganisms and virusesspecifically. Many airborne pathogens such as rhinoviruses and influenzagain entry into nasal epithelial cells, nasal mucosa, or cells of thelower respiratory tract through specific cell surface targets. Decadesof research have identified ICAM-1 (Intracellular Adhesion Molecule-1)as one such target for most rhinoviruses and another for influenza(Abraham and Colonno 1984). ICAM-1 is an intercellular adhesion moleculeexpressed on the cell surface of nasal epithelial cells, as well ascells of the lower respiratory tract. The N-terminal domain of ICAM-1 isrecognized by receptors on certain rhinovirus capsids. Upon bindingICAM-1, the virus sheds its capsid and is transported into the cellwhere it initiates infection and an inflammatory response by the host.Influenza viruses exhibit a similar mechanism of infection: in humans,hemagluttinin (HA) on viral surfaces bind sialic acid attached togalactose (e.g. by an alpha 2,6 linkage (6′-sialyllactose) or by analpha 2,3 linkage (3′-sialyllactose)), on the host cell membrane oferythrocytes and cells of the upper respiratory tract.

Prior art such as, for example, U.S. Pat. Nos. 8,211,448, 8,940,339, and8,211,448 disclose methods of entrapping airborne particles by usingmanufactured polymers or compounds. In sharp contrast to these prior artmethods, the present disclosure renders airborne particles inert andaugments the abilities of the nasal membranes to eliminate disease orallergy causing microorganisms and other undesirable particlesthemselves. The present disclosure also differs from the prior art inhaving more than one active ingredient such that the techniques hereinare able to protect against more than one pathogen or irritant at thesame time and in an augmented fashion. This differs from prior artmethods that may target only one type of pathogen only weakly ormoderately (e.g., U.S. Pat. No. 7,132,395; 6,514,936; 6,051,231;6,649,592; Turner et al. JAMA 281 (19). 1797-1804. (1999)). This isimportant because the specific identity of a disease or allergy causingmicroorganism is often unknown at the time of initial exposure to asubject. The present disclosure does not intend to deliver drugs to thenasal membranes or to be a bioadhesive as does some prior art (e.g.,U.S. Pat. No. 6,391,452; US Publication No. 2001/0053359; U.S. Pat. No.8,679,484; U.S. Pat. No. 6,456,26; U.S. Pat. No. 7,087,245). The presentdisclosure also differs from prior art that imparts a protective layerwith broad-based antimicrobial properties (e.g., US Publication No.2007/0135377; U.S. Pat. Nos. 7,166,435; 8,658,775; 8,658,775; 7,083,814;7,807,656; 9,045,855; 6,649,592; 9,029,351) because the techniquesdisclosed herein add specifically targeted antimicrobials that work in asynergistic manner with other ingredients to maintain the specificphysiological and chemical properties of the nasal membranes or mucoussuch as, for example, their pH and osmolarity. Similarly, the presentinvention does not intend to add non-targeted, indiscriminantantimicrobials to the nasal membranes that may contain alcohol,peroxide, or other harsh ingredient that would change the pH orosmolarity of the nasal membranes or negatively impact microflora asdoes some prior art (e.g., U.S. Pat. Nos. 8,999,406; 8,778,415;7,638,147).

Antimicrobial Compositions

The antimicrobial compositions herein may incorporate one or moreantimicrobial and antiviral active ingredients within a base mixturecomprising one or more of water, polyols, emollients, occlusives,humectants, emulsifiers, preservatives, thickeners and suspendingagents, pH adjusters, isotonicity agents, and essential oils that allowit to remain at or near the site of application for at least 30 minutes(e.g. 30-60 minutes, 1-2 hours, 2-4 hours, 4-8 hours, 8-12 hours, 12-24hours, 1-2 days, 2-7 days, a week or more) before being absorbed, andwhich have similar pH and osmolarity to mucous, and which do not clogpores. The time it takes for the antimicrobial composition to beabsorbed can be determined by the saccharine test or other similartests. In one embodiment, the active ingredient may be soluble ICAM-1,comprising just the extracellular domain, or another domain of ICAM-1recombinantly expressed in bacteria. In other embodiments, the activeingredient may be soluble ICAM-1 recombinantly expressed inChlamydomonas reinhardtii. In other embodiments, the active ingredientmay be soluble ICAM-1 recombinantly expressed in another species ofalgae, or another living system.

In another embodiment, the active ingredient is sialic acid (e.g.,neuraminic acid linked to a sugar molecule in one of several possibleconformations). The sialic acid may be purified from a natural source orproduced by fermentation and recombinant engineering. In anotherembodiment, the active ingredient may be a neuraminidase inhibitor suchas quercetin, which is a bioflavanoid isolated from citrus peels orother natural sources. In another embodiment, the active ingredient islactoferrin recombinantly expressed in bacteria. In other embodiments,the active ingredient is lactoferrin recombinantly expressed inChlamydomonas reinhardti or another appropriate expression system, e.g.algae, yeast, or bacteria, or purified from a natural source. In anotherembodiment, the active ingredient may be lysozyme recombinantlyexpressed in Chlamydomonas reinhardti or another appropriate expressionsystem, e.g. algae, or purified from a natural source. As disclosedherein, additional antimicrobial active ingredients may be used in thecompositions of the disclosure, either alone or in combination.

In another embodiment, the active ingredient is a naturally occurring orgenetically engineered antibody such as IgA, IgG, or IgM, or any oftheir domains in one of several possible conformations. The antibody maybe purified from a natural source or produced by recombinant engineeringin any appropriate and economically feasible expression system such asChlamydomonas reinhardti or another algae, a bacteria, a yeast, or amammalian cell, or it may be purified from a natural source. In anotherembodiment, the active ingredient may be a neuraminidase inhibitor suchas quercetin, which is a bioflavanoid isolated from citrus peels orother natural sources.

Algae or other plants are the preferred expression system becauserecombinantly engineered algae are far more economical to grow andharvest than mammalian cells or bacteria. Algae are grown for use asnutritional supplements themselves and are also used to bioengineercertain nutritional compounds for commercial production such as omega-3fatty acids and carotenoids (Gimpel J A, Henriquez V, and Mayfield S PFrontiers in Microbiology 2015). Most metabolic engineering strategieshave been geared towards enhancing commercial production of thesecompounds and also for using algae as biofuels. Advantageously, algaemay be optimized to produce a range of different metabolites that havecertain characteristics in an efficient manner. The expression of activeingredients may be optimized.

Transgenic algae have been shown to support recombinant proteinexpression from both the chloroplast and nuclear genomes (Rasal B A etal., Plant Biotechnology J 2010). Originally, only the nuclear genomeswere used but the development of techniques required to expressrecombinant proteins in the chloroplast genome add versatility to theplatform and make it possible to either express proteins that cannot beexpressed in the nuclear genome or to express the proteins moreefficiently. The majority of recombinant proteins produced today areproduced mainly in bacteria, yeast (S. cervisiae), or mammalian cellculture. Other systems under development for large scale productioninclude the yeast P. pastoris, insect cells, and other animals andplants. Any viable plant or animal expression system may be used butfirst, those which are likely to be the most cost-efficient such asthose recombinant expression systems that will not require a high degreeof purification will be investigated and sought out. This will make itpossible for the embodiment to be sold over the counter without the needfor clinical trials. If needed, other recombinant expression systems areused that may require higher degrees of purification.

As depicted in Table 1, the agents of the composition may be presentaccording to the following percentages by weight of the composition:

TABLE 1 Example formulation Active Ingredients % by weight (% by weight)sICAM-1 0.00000001%-10.0% 0.000001%-1% Lactoferrin 0.00000001%-10.0%0.000001%-1% Lysozyme 0.00000001%-10.0% 0.000001%-1% Sialic acid0.00000001%-10.0% 0.000001%-1% Neuraminidase inhibitor 0.00000001%-10.0%0.000001%-1%

As indicated in Table 1, a neuraminidase inhibitor is optional but maybe desirable in some embodiments, particularly where the composition isintended for prophylaxis or treatment of human influenza viralinfection. The sialic acid may be in the form of free sialic acid, ormay be the conjugate or adduct of sialic acid with a saccharide such asgalactose, lactose, etc. Sialic acid may be any sialic acid for exampleacids within the sialic acid family which includes at least 43derivatives conjugates or adducts of the nine-carbon sugar neuraminicacid. What is important is that the sialic acid, in whatever form, becapable of binding the influenza virions. Furthermore, the lactoferrinshown in Table 1 may be any form of lactoferrin including lactoferrinsfree from chelation with iron (apolactoferrin), lactoferrins rich iniron (hololactoferrin) or combinations thereof. As used herein, “ICAM-1”includes any form of ICAM-1 including, without limitation, theextracellular domain portions of ICAM-1, and, in particular, solubleICAM-1 (“sICAM-1”). It will be understood that in any of thecompositions of the invention which call for ICAM-1, soluble ICAM-1 maysuitably be included.

As depicted in Table 1, ingredients may be present in a range of percentweights of the composition. ICAM-1 (including soluble ICAM-1) may bepresent in an amount from about 0.00000001% to 10% by weight ofcomposition. More typically, ICAM-1 (including soluble ICAM-1) may bepresent in an amount from 0.0000001% to 0.1% by weight of thecomposition. More typically, ICAM-1 (including soluble ICAM-1) may bepresent in an amount from 0.000001% to 0.01% by weight of thecomposition. ICAM-1 may be present in an amount of about 0.00000001%,0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.05%, 1%,0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%,0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.05%, 1.1%, 1.15%,1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%,1.76%, 1.8%, 1.85%, 1.9%, 1.95%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%,5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, and 10.0% byweight of composition. In some embodiments, ICAM-1 may be present from0.2% to 1.0% by weight of composition. In some embodiments, ICAM-1 maybe present from about 0.000005% to 0.05% by weight of the composition.In preferred embodiments, ICAM-1 may be present from about 0.00005% to0.005% by weight of the composition.

A neuraminadase inhibitor (e.g., quercetin, etc.) may be present in anamount from about 0.00000001% to 10% by weight of composition. Moretypically, neuraminidase inhibitor may be present in an amount from0.0000001% to 0.1% by weight of the composition. More typically, aneuraminidase inhibitor may be present in an amount from 0.000001% to0.01% by weight of the composition. A neuraminadase inhibitor may bepresent in an amount of about 0.000000001%, 0.0000001%, 0.000001%,0.00001%, 0.0001%, 0.001%, 0.01%, 0.05%, 1%, 0.1%, 0.15%, 0.2%, 0.25%,0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%,0.85%, 0.9%, 0.95%, 1.0%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%,1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.76%, 1.8%, 1.85%, 1.9%,1.95%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%,7.5%, 8.0%, 8.5%, 9.0%, 9.5%, and 10.0% by weight of composition. Insome embodiments, a neuraminidase inhibitor may be present from 0.2% to1.0% by weight of composition. In some embodiments, a neuraminadaseinhibitor may be present from about 0.000005% to 0.05% by weight of thecomposition. In preferred embodiments, a neuraminadase inhibitor may bepresent from about 0.00005% to 0.005% by weight of the composition.

Lactoferrin may be present in 0.0000001% to 10.0% by weight of thecomposition. Lactoferrin may be present in 0.00000025%, 0.0000003%,0.0000004%, 0.0000005%, 0.0000006%, 0.0000007%, 0.00000075%, 0.0000008%,0.0000009%, 0.000001%, 0.000002%, 0.000003%, 0.000004%, 0.000005%,0.000006%, 0.000007%, 0.000008%, 0.000009%, 0.00001%, 0.00002%,0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%,0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008,0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%,0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%,0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%,2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0% 6.5%, 7.0%, 7.5%, 8.0%,8.5%, 9.0%, 9.5%, and 10.0% by weight of composition. In preferredembodiments, lactoferrin may be present from 0.0000001-1.0% by weight ofcomposition.

Lysozyme may be present in 0.000001% to 10.0% by weight of thecomposition. Lysozyme may be present in 0.000005%, 0.000006%, 0.000007%,0.000008%, 0.000009%, 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%,0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%,0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008, 0.0009%, 0.001%, 0.002%,0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%,0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%,0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%,4.5%, 5.0%, 5.5%, 6.0% 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, and10.0% by weight of composition. In preferred embodiments, lysozyme maybe present from 0.000001-1.0% by weight of composition.

Sialic acid may be present in 0.000000001% to 10.0% by weight of thecomposition. Sialic acid may be present in 0.000005%, 0.000006%,0.000007%, 0.000008%, 0.000009%, 0.00001%, 0.00002%, 0.00003%, 0.00004%,0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%,0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008, 0.0009%, 0.001%,0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%,0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%,3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0% 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%,9.5%, and 10.0% by weight of composition. In preferred embodiments,sialic acid may be present from 0.000001-1.0% by weight of composition.Sialic acid may be in the form of sialyllactose (e.g., 3′-sialyllactose,6′-sialyllactose, or combinations thereof).

In another embodiment, a formula may have the ingredients as shown inTable 2.

TABLE 2 Amount (% by weight of Active Ingredients composition) sICAM-10.00000001%-10.0% Lactoferrin 0.00000001%-10.0% Lysozyme0.00000001%-10.0% Sialic Acid 0.00000001%-10.0% neuraminisade inhibitor0.00000001%-10.0% IgA, IgG, IgM  0%-0.00000001%-10% Zinc (ZnO₂)0%-0.00000001%-5% Copper 0%-0.00000001%-5% Silver 0%-0.00000001%-5%

Carageenan may be present in 0.0000005% to 10.0% by weight ofcomposition. Lysozyme may be present in 0.0000005%, 0.0000006%,0.0000007%, 0.0000008%, 0.0000009%, 0.000001%, 0.000002%, 0.000003%,0.000004%, 0.000005%, 0.000006%, 0.000007%, 0.000008%, 0.000009%,0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%,0.00008, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%,0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%,0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%,0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%,6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, and 10.0% by weight ofcomposition. In preferred embodiments, carageenan may be present from0.000001-4.0% by weight of composition.

Zinc (e.g. zinc peroxide) may be present in 0.0000001 to 5% by weight ofcomposition. Zinc may be present in 0.01%, 0.015%, 0.02%, 0.025%, 0.03%,0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.060%, 0.065%, 0.070%, 0.075%,0.080%, 0.085%, 0.090%, 0.095%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%,0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%,0.95%, 1.0%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%,1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.76%, 1.8%, 1.85%, 1.9%, 1.95%, 2.0%,2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%,8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%,13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%,18.5%, 19.0%, 19.5%, and 20.0% by weight of composition. In preferredembodiments, zinc peroxide (ZnO₂) may be present from 0.000001%% to 5.0%by weight of composition.

Copper may be present in 0.00000001% to 5% by weight of composition.Copper may be present in 0.000005%, 0.000006%, 0.000007%, 0.000008%,0.000009%, 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%,0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%,0.0005%, 0.0006%, 0.0007%, 0.0008, 0.0009%, 0.001%, 0.002%, 0.003%,0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%,0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%,5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5% 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%,11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%,16.0%, 16.5%, 17.0%, 17.5%, 18.0% 18.5%, 19.0%, 19.5%, and 20.0% byweight of composition. In preferred embodiments, copper may be presentfrom 0.00001-5.0% by weight of composition.

Silver may be present in 0.00000001% to 5% by weight of composition.Silver may be present in 0.000005%, 0.000006%, 0.000007%, 0.000008%,0.000009%, 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%,0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%,0.0005%, 0.0006%, 0.0007%, 0.0008, 0.0009%, 0.001%, 0.002%, 0.003%,0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%,0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%,5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%,10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%,15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0% 18.5%, 19.0%, 19.5%, and 20.0%by weight of composition. In preferred embodiments, silver may bepresent from 0.00001-5.0% by weight of composition.

The composition may also include odor-neutralizing compounds such asactivated charcoal or sodium bicarbonate alone or in combination. Otherodor-neutralizing compounds are contemplated for inclusion in thecomposition.

As depicted in Table 3, an exemplary antimicrobial composition mayfurther include the following additional ingredients, either alone or incombination: marshmallow extract, calendula extract, citrus peelextract, honey, rosemary extracts, myrhh extract, helichyrsum extract,arrowroot extract, neem oil, vitamin C, vitamin E, and grapefruit seedextract. These additional ingredients may be present according to thefollowing weights of the composition. The extraction solvents andextraction processes will be optimized for the antimicrobial compositionto be most effective and tolerable.

TABLE 3 Additional Ingredients Supportive or Herbal Amount (% by weightof Ingredients composition) Marshmallow extract 0.00000001%-5% Calendula extract 0.00000001%-10% Citrus peel extract 0.00000001%-10%Honey extracts 0.00000001%-10% Rosemary extracts 0.00000001%-10% Myrhhextract 0.00000001%-10% Helichrysum extract 0.00000001%-10% Arrowrootextract 0.00000001%-10% Neem oil 0.00000001%-10% Vitamin C0.00000001%-10% Vitamin E 0.00000001%-10% Grapefruit seed extract0.00000001%-10% Activated charcoal 0.00000001%-10% Sodium bicarbonate0.00000001%-10%

As depicted in Table 4, an exemplary antimicrobial composition mayfurther include the following additional base ingredients either aloneor in combination: a polyol, a humectant (such as but not limited toglycerine, aloe vera, or butylene glycol), an emmolient (such as but notlimited to shea butter, castor oil, coconut oil, caprilic acid, butylstearate, or triglyceride), an occlusive substance (such as but notlimited to petroleum jelly, dimethicone, lanolin, cocoa butter, sheabutter, beeswax, plant butters, or carnauba wax). The addition and exactconcentrations of these ingredients may be optimized to achieve abarrier function that does not clog pores, keeps active ingredients intheir bioactive conformations and in place for at least 30 minutes,maintains osmolarity and pH similar to physiological mucus, is tolerableand effective when applied.

TABLE 4 Additional Base Ingredients Base Amount (% by weight ofIngredients composition) Humectant 0.01%-10%-75% Emollient 0.01%-10%-75%Occlusive 0.01%-10%-75% Preservatives 0.00001%-0.5%-5% Emulsifiers0.01%-1%-20% Thickener 0.001%-1%-10%

Unless otherwise indicated, any excipient or additive may be included inan amount sufficient to serve its intended functional purpose withoutharming or irritating the respiratory tissues. Unless otherwiseindicated, all excipients may be present in an amount from about0.000001% or 0.01% to about 1, 5, 10, or 25% by weight of thecomposition. The carrier may be any pharmaceutically acceptable diluentand may be solid at room temperature or liquid at room temperature.Suitable carriers include water, alcohol (ethanol), Propylene glycol,isopropanol, propanediol, glycerin, benzyl alcohol, isostearlyisosterate, caprylic/capric triglyceride, oleyl oleate, tocopherolacetate, decyl cocoate, squalene, span 40, coco-caprylate/caprate, span80, tocopherol, osstearic acid, oleyl erucate, span 20, glycerylisostearate, and caprylic/capric triglyceride. The carrier may be, forexample, in the form of an aqueous solution, a water in oil emulsion, oran oil in water emulsion. The emulsion carrier will typically comprisefrom 0.0001% to about 10% by weight of an emulsifier suitable tostabilize the emulsion. In one embodiment, the composition is intendedfor oral use and may include one or more of the following ingredients:glucose syrup, soy lecithin, alginates, sucralose, corn syrup, gelatin,erythritol, lecithin, plant mucins, carrageenan, chicory root extract,malitol, and stevia. In both oral and nasal formulations, it should benoted that the excipients should not interfere with the biologicalactivity of the actives and should be compatible in solution with theactives. The excipients should also not penetrate the epithelia(respiratory membranes). Accordingly, it is desirable to have a highersolubility of the actives in the base composition to maintain them insolution and prevent penetration of the actives into the epithelia. Innasal formulations, the excipients should serve to maintain moisture inthe mucosa and the compositional film thereon, such that the activeingredients do not precipitate from solution. In some embodiments, thecarrier will comprise water, and a secondary solvent of lower volatilitythan the water in which the actives are also soluble. Ideally, thecarrier and excipients are selected such that they have a polarity thatis closer to the polarity of the active ingredients than to the polarityof the epithelia. The secondary solvent may serve to keep the activessoluble after water has evaporated. In oral formulations, loss ofmoisture is less important and it may be desirable to select excipientsso as to retard the dissolution of the composition in the saliva.

Prepared compounds are purified using conventional methods to obtaincompounds free of impurities. Prepared compoundsare >75%, >80%, >85%, >90%, >95%, >96%, >97%, >98%, >99%, >99.5% pure.Optionally, preferred compounds are >99% pure. Intracellular AdhesionMolecule (ICAM-1)

ICAM-1 is a member of the immunoglobulin superfamily of adhesionmolecules. It is an integral membrane protein 505 amino acids long andhas: i) five immunoglobulin-like extracellular domains at theamino-terminal (extracellular) end, ii) a hydrophobic transmembranedomain (454-477); and iii) a short cytoplasmic domain at thecarboxy-terminal end (478-505). Most rhinoviruses use ICAM-1 expressedon nasal epithelial membranes to gain entry into cells. There are threemain types of rhinoviruses (HRV A, B, and C). HRV A and B can be furthersubdivided into about 100 subtypes. 85% of HRVA and 100% of HRVB useICAM-1 to enter cells. HRV C rarely causes noticeable illness. Accordingto the techniques herein, the addition of partially or fully purifiedsoluble forms of ICAM-1, or any of its recombinantly engineered domains,to the compositions of the disclosure may keep the molecule in a stableform whereby it can competitively inhibit rhinovirus particles frominteracting with endogenous ICAM-1 on cell membranes, thus preventingthe first step of infection.

In some embodiments, the antimicrobial/antiviral compositions may becomprised of ICAM-1 inhibitors. ICAM-1 inhibitors used in theantimicrobial compositions include, but are not limited to, ananti-ICAM-1 antibody, cytokine, CD11a, ezrin (EZR), CD18, glycyrrhetinicacid, pyrrolidinedithiocarbamate, NFkB activation inhibitor,heterocyclic thiazole, lipoic acid, efalizumab,4-[(4-Methylphenyl)thio]thieno[2,3-c]pyridine-2-carboxamide, silibinin,stilbenes, (+)-epigalloyl-catechin-gallate [(+)-EGCG], extracts of Pipersarmentosum, and combinations thereof. In some embodiments, theanti-ICAM-1 antibody is efalizumab (RAPTIVA).

Sialic Acid and Neuraminidase Inhibitors

Sialic acid rich oligosaccharides on cells of the upper respiratorytract normally help keep water on the surface and create a negativecharge. Sialic acid is a generic term for the N- or O-substitutedderivatives of neuraminic acid, a monosaccharide with a nine-carbonbackbone. It is also the name for the most common member of this group,N-acetylneuraminic acid (Neu5Ac or NANA). Influenza or other viruses areknown to bind sialic acid residues via the hemagglutinin (HA) receptoron their surface after which they begin to replicate. When replicationis complete, a viral enzyme, neuraminidase cleaves the viral particlesso they are free to bind and infect other cells. According to thetechniques herein, partially or fully purified sialic acid such as, e.g.N-acetylneuraminic acid (Neu5Ac), or any other neuraminic acidderivative or any of their recombinantly engineered domains may be addedto keep the molecule in a stable form in which it may competitivelyinhibit influenza virus particles from interacting with sialic acid onrespiratory cells or erythrocytes. The sialic acid family includes 43derivatives of the nine carbon sugar neuraminic acid. In nature, theyare usually found as components of oligosacharride chains of mucins,glycoproteins, and glycolipids. Various species susceptible to influenzaare thought to have slight variations in their sialic acid-galactoselinkages. Influenza viruses that infect a particular species are thoughtto have specific affinity for sialic acid bound to galactose in thatspecies-specific conformation. For example, on human respiratoryepithelial cells sialic acid is primarily attached to galactose via analpha 2,6 linkage so influenza viruses that infect humans are usuallyspecifically targeted to that conformation of sialic acid. Humanepithelial cells also have other less predominant types of sialicacid-galactose linkages such as the alpha 2,3 linkage that may be thepredominant conformation in another species such as pigs. As such, onetheory of how influenza might spread from one species to another is byhaving specificity for a conformation that is found in more than onespecies, or in mutating to become specific for another one.

Nasal mucous and other exocrine secretions contain sialic acid insoluble form which may serve a protective function by binding influenzaviruses and other microorganisms and immobilizing them. Influenzaviruses have evolved a method for cleaving themselves from solublesialic acid using an enzyme called neuraminidase. Several antiviraldrugs such as Tamiflu serve as neuraminidase inhibitors. In this regard,they keep the virus immobilized and unable to infect epithelial cells.There is no prior art attempting to use neuraminidase inhibitors in atopical solution applied to the nostrils to prevent infection. It ismainly administered systemically to cure infection. Quecertin, abioflavanoid found in citrus peels and many other fruits and vegetablesis an example of a naturally occurring neuraminidase inhibitor.

In some embodiments, antimicrobial compositions include neuraminidaseinhibitors. Neuraminidase inhibitors include, but are not limited to,quercetin, oseltamivir, zanamivir, laninamivir, amantadine, peramivir,and any of their analogues.

Anti-Viral Agents

The anti-viral agents of the present invention may be obtained bynatural processes (e.g., by inducing an animal, plant, fungi, bacteria,etc., to produce an analog of ICAM-1, or by inducing an animal toproduce polyclonal or monoclonal anti-ICAM-1 anti-idiotypic); syntheticmethods (e.g., by using the Merrifield method for synthesizingpolypeptides of a functional derivatives of ICAM-1, etc.); recombinanttechnology (e.g., to produce the anti-viral functional derivatives ofICAM-1 in diverse hosts (e.g., yeast, bacteria, fungi, culturedmammalian cells, etc.), recombinant plasmids or viral vectors, orproteolysis. The choice of which method to employ depends upon factorssuch as convenience, desired yield, etc. It is not necessary to employonly one of the above-described methods, processes, or technologies toproduce a particular anti-viral agent; the above-described processes,methods, and technologies may be combined in order to obtain aparticular anti-viral agent. Ingredients are balanced to createsynergistic effects.

Lactoferrin

Lactoferrin is naturally present in exocrine secretions including nasalmucous and serves a protective function against microorganisms. It ishighly cationic, anti-bacterial, anti-viral, and anti-fungal. In tears,concentrations range from 1-3 mg/ml accounting for 15-30% of totalprotein. Human milk contains 1 mg/ml and bronchial secretions can haveas much as 11.5% total protein. Concentrations in mucous range from 1μg/ml to 8 μg/ml with challenge and from about 1-3% total protein(Raphael et al 1989). Levels of lactoferrin in mucosal secretions arethought to decrease with age, which might make older adults moresusceptible to respiratory infection.

Lactoferrin exists in monomeric and tetrameric forms and tends topolymerize at high concentrations. It is a glycoprotein of approximately703 amino acids and approximately 80 kD. Despite a 69% homology betweenhuman lactoferrin and bovine lactoferrin, in some studies, bovinelactoferrin was required at 1/10 the concentration of human lactoferrinfor an antimicrobial effect. Different types of lactoferrin may be morebeneficial for certain mutations or types of infective organisms thanothers and can be tested to be optimized in the case of pendingepidemics. In studies of herpes simplex virus (topical application) andhepatitis C infection, lactoferrin was protective before contact withthe virus but not after, making application to the site of infection(such as nasal membranes) more relevant than systemic application.

Lysozyme

Lysozyme, like lactoferrin, is normally present in exocrine secretions.It is also highly cationic, anti-bacterial, anti-viral, and anti-fungal.According to one study (Atsushi et al 1998), the average concentrationin mucous is 20-30 μg/ml. In tears, the concentration of lysozyme isabout 103 mg/ml according to Raphael et al 1989. Levels of lysozyme inmucous are also thought to decrease with age.

Mucosal Antibodies

Nasal secretions contain immunoglobulins offering antibody mediateddefense and research indicates a majority is the secretory form of IgA(sIgA) (Kirkeby et al., 2000). S-IgA antibodies prevent microbialattachment and the absorption of molecular antigens including potentialallergens. Certain bacteria produce IgA proteases, by cleaving IgA,these enzymes may interfere with the barrier function of theseantibodies. Research indicates that cleavage of IgA may result in atopicdisease. Other antibodies most commonly found in nasal secretions andwhich may serve protective funtions are IgG and IgM (Kirkeby et al2000). By augmenting the amounts of these antibodies, the presentdisclosure may protect against undesirable irritants or pathogens.

Recombinant Engineering

Several possible sources of the aforementioned biological compoundsexist such as human or bovine exocrine secretions. However, these may bein limited supply and pose safety risks. Recombinant bio-manufacturingis another possible source of the compounds. Bio-manufacturing canutilize genetically engineered microorganisms like bacteria, fungi,animal cells, yeast, or plants (including algae). Expression inmammalian cell lines, bacteria, and yeast is often costly. One of thereasons for this is the need for purification. Algae offer severaladvantages to other methods in that very high levels of purification areoften not required. It is estimated that protein production in plantscan be as much as four orders of magnitude less expensive thanproduction in mammalian cell culture on a per gram of unpurified proteinbasis. In addition, plant material such as algae is for the most part“Generally Regarded as Safe” as are their genetically modifiedcounterparts. Commercial scale production seems feasible sincerecombinant algal bioreactors for several classically expensivebiological molecules has proven promising (see Rasala et al PlantBiotech. 2010).

Methods of Treatment

Novel methods and compositions for enhancing the filtering capabilitiesof the respiratory membranes and protecting against airborne pathogensare described herein. The delivery methods of the present disclosuremaximize exposure of the airway to antimicrobial/antiviral compositionsfor protection against airborne pathogens. The novel therapeutic methodsmay also involve administration of an antimicrobial composition as atherapeutic agent.

In any of the above aspects or embodiments, the method may reduce thegrowth of a respiratory infection, shrink the infection, or eradicatethe infection. In related embodiments, the infection shrinks by 5%, 10%,25%, 50%, 75%, 85%, 90%, 95%, or 99% or more as compared to its originalsize.

In any of the above aspects or embodiments, the methods may involveadministering the therapeutic agent multiple times per day. In yetfurther related embodiments, the methods may involve administering thetherapeutic agent on a first day and repeating the administration on oneor more subsequent days. In yet further related embodiments, the firstday and one or more subsequent days are separated by between 1 day andabout 3 weeks. In related embodiments, the therapeutic agent and anotheragent are coadministered. In related embodiments, the therapeutic agentand other agents are administered in a ratio of about 1:2, 1:4, 1:10,1:20, 1:25, 1:50, 1:100, 1:200, or any ratio there between (weight ratioof therapeutic agent:agent). It is further contemplated within the scopeof the disclosure that the therapeutic agent may be administered overthe course of one or more cycles. In any of the above aspects orembodiments, the therapeutic agent and another agent can be deliveredsimultaneously.

In any of the above aspects or embodiments, the therapeutic agent may beany antimicrobial composition as described herein that is used toprevent or treat a respiratory disease or disorder. In relatedembodiments, the respiratory disease or disorder is caused by airbornepathogens. In certain embodiments, the therapeutic agent is anantimicrobial/antiviral agent.

The antimicrobial agent can be any agent well known in the art,including, but not limited to, those described herein.

In yet other embodiments, the therapeutic agent may be a therapeuticantibody. The therapeutic antibody can be any therapeutic antibody wellknown in the art, including, but not limited to, those described herein.

In embodiments, the therapeutic agent may be a therapeutic nucleic acidmolecule. The therapeutic nucleic acid molecule can be any therapeuticnucleic acid molecule well known in the art.

In embodiments, the therapeutic agent may be a radioisotope. Theradioisotope may be any radioisotope well known in the art.

In other embodiments, the therapeutic agent may be a combination of twoor more drug compounds.

In any of the above aspects or embodiments, the methods involveadministering a therapeutically effective amount of an immunotherapeuticagent. The immunotherapeutic agent may be any suitable means by which totrigger a further immune response that targets destruction of the cellsof the infection.

In embodiments, the immunotherapeutic agent enhances theimmunomodulatory effects of the therapeutic agent. In relatedembodiments, the immunotherapeutic agent further reduces the growth ofthe infection or further shrinks the infection.

The immunotherapeutic agent may be administered before, during, or afterthe therapeutic agent has been administered. In embodiments, theimmunotherapeutic agent is administered before the first administrationof the therapeutic agent. In embodiments, the immunotherapeutic agent isadministered simultaneously with the first administration of thetherapeutic agent.

In any of the above aspects or embodiments, the therapeutic agent andthe immunotherapeutic agent can be administered in a ratio of about 1:2,1:4, 1:10, 1:25, 1:50, 1:100, 1:200, or any ratio there between (weightratio of therapeutic agent:immunotherapeutic agent).

In any of the above aspects or embodiments, the immunotherapeutic agentcan be administered intranasally, locally, regionally, or systemically(e.g. intravenously).

In any of the above aspects or embodiments, the therapeutic agent andthe immunotherapeutic agent may be coupled.

Therapeutic Agents

The present disclosure contemplates any therapeutic agent suitable foruse in the methods described herein (e.g., any type ofantimicrobial/antiviral agent to treat respiratory disease). Suitabletherapeutic agents include, but are not limited to, pharmaceutical drugsor compounds (i.e., small molecule drugs), therapeutic antibodies,therapeutic proteins or biologics (e.g., hormone therapies), and nucleicacid molecules (e.g., siRNAs).

In some embodiments, the therapeutic agent is an agent that has beenshown to have antimicrobial properties against infectious organisms. Inrelated embodiments, the therapeutic agent is an existingmarket-approved pharmaceutical drug or other market-approved compositionfor treating infection using a conventional approach.

In some embodiments, the therapeutic agent is an antimicrobialcomposition as described herein. In some embodiments, antimicrobialcompositions include compositions with anti-bacterial, anti-viral,and/or anti-fungal properties. Antimicrobial compositions include butare not limited to: an antibody such as IgA, IgG, or IgM, a solubleICAM-1, an ICAM-1 inhibitor, sialic acid, a neuraminidase inhibitor,lactoferrin, a lysozyme, a zinc compound, silver, silver compounds,copper, copper compounds, and combinations thereof. Neuraminidaseinhibitors include but are not limited to: quercetin, oseltamivir,zanamivir, laninamivir, and peramivir. ICAM-1 inhibitors include but arenot limited to: soluble ICAM-1, an anti-ICAM-1 antibody, cytokine,CD11a, ezrin (EZR), CD18, glycyrrhetinic acid,pyrrolidinedithiocarbamate, NFkB activation inhibitor, heterocyclicthiazole, lipoic acid, efalizumab,4-[(4-Methylphenyl)thio]thieno[2,3-c]pyridine-2-carboxamide, silibinin,stilbenes, (+)-epigalloyl-catechin-gallate [(+)-EGCG], and combinationsthereof. In some embodiments, the anti-ICAM-1 antibody is efalizumab(RAPTIVA).

The disclosure also contemplates any derivative form of theaforementioned pharmaceutical agents and therapeutic agents. Commonderivatizations may include, for example, adding a chemical moiety toimprove solubility and/or stability, or a targeting moiety, which allowsmore specific targeting of the molecule to a specific cell or region ofthe body. The pharmaceutical agents may also be formulated in anysuitable combinations, wherein the drugs may either mixed in individualform or coupled together in a manner that retains the functionality ofeach drug. The drugs may also be derivatized to include a radioisotopeor other cell-killing moiety to make the molecule even more effective atkilling the cell. In addition, the drugs, or a portion thereof, may bemodified with fluorescence compound or other detectable labels which mayallow tracking of the drug or agent in the body or within the tumor. Thepharmaceutical drug or otherwise any of the aforementioned therapeuticagents may be provided in a precursor form such that they the drug onlygains its activity or function after it has been processed in somemanner, e.g., metabolized by a cell.

Therapeutic antibodies contemplated by the present disclosure mayinclude any isotype (IgA, IgG, IgE, IgM, or IgD) of an anti-microbial orantiviral antibody or immune-active fragment or derivative thereof. Suchfragments may include, for example, single-chain variable fragments(scFv), antigen-binding fragment (Fab), crystallizable fragment (Fc)modified to contain an antigen or epitope binding region, and domainantibodies. Derivatized versions of therapeutic antibodies may include,for example, diabodies, nanobodies, bispecific antibodies, and virtuallyany antibody-derived structure which contains or is engineered tocontain an appropriate and effective antigen binding site.

Examples of antibody-based antimicrobial therapies that may be utilizedby the disclosure can include, for example, an antibody specific forICAM-1. In some embodiments, the anti-ICAM-1 antibody is efalizumab(RAPTIVA).

The disclosure also contemplates that preventing or treating respiratorydisease may be effected using a nucleic acid molecule that targets aspecified “target gene” that has a role in infection. The effect of thenucleic acid molecule on the target gene may include gene silencing,mRNA destruction, or inhibited transcription, or the like, such that thelevel of expression and/or conversion of the target gene to an operableencoded polypeptide are substantially affected (up or down) such thatthe cancer is inhibited and/or destroyed by the agent. The term “targetgene” refers to nucleic acid sequences (e.g., genomic DNAs or mRNAs)encoding a target protein, peptide, or polypeptide, or that encode foror are regulatory nucleic acids (e.g., a “target gene” for purpose ofthe instant disclosure can also be a miRNA or miRNA-encoding genesequence) which have a role in infection. In certain embodiments, theterm “target gene” is also meant to include isoforms, mutants,polymorphisms, and splice variants of target genes.

Any nucleic acid based agent well known in the art may be suitable foruse in the disclosure. Exemplary types of nucleic acid based agentsinclude, but are not limited to, single stranded ribonucleic acid agents(e.g., microRNAs), antisense-type oligonucleotide agents,double-stranded ribonucleic acid agents, and the like.

Methods for constructing therapeutic nucleic acids are well known in theart. For example, interfering RNA can be assembled from two separateoligonucleotides, where one strand is the sense strand and the other isthe antisense strand, wherein the antisense and sense strands areself-complementary (i.e., each strand comprises a nucleotide sequencethat is complementary to nucleotide sequence in the other strand; suchas where the antisense strand and sense strand form a duplex or doublestranded structure); the antisense strand comprises nucleotide sequencethat is complementary to a nucleotide sequence in a target nucleic acidmolecule or a portion thereof and the sense strand comprises nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof.

Alternatively, interfering RNA may be assembled from a singleoligonucleotide, where the self-complementary sense and antisenseregions are linked by means of nucleic acid based or non-nucleicacid-based linker(s). The interfering RNA may be a polynucleotide with aduplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises a nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. The interfering RNA can be a circular single-strandedpolynucleotide having two or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense region having nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof, and wherein the circularpolynucleotide can be processed either in vivo or in vitro to generatean active siRNA molecule capable of mediating RNA interference.

Methods for administering/delivering therapeutic nucleic acids are wellknown in the art. For example, therapeutic nucleic acid molecules may bedelivered in a delivery vehicle, such as a lipid vesicle or otherpolymer carrier material known in the art. Non-limiting examples ofadditional lipid-based carrier systems (which may be prepared with atleast one modified cationic lipid of the disclosure) suitable for use inthe present disclosure include lipoplexes (see, e.g., U.S. PatentPublication No. 20030203865; and Zhang et al., J. Control Release,100:165-180 (2004)), pH-sensitive lipoplexes (see, e.g., U.S. PatentPublication No. 2002/0192275), reversibly masked lipoplexes (see, e.g.,U.S. Patent Publication Nos. 2003/0180950), cationic lipid-basedcompositions (see, e.g., U.S. Pat. No. 6,756,054; and U.S. PatentPublication No. 2005/0234232), cationic liposomes (see, e.g., U.S.Patent Publication Nos. 2003/0229040, 2002/0160038, and 2002/0012998;U.S. Pat. No. 5,908,635; and PCT Publication No. WO 01/72283), anionicliposomes (see, e.g., U.S. Patent Publication No. 2003/0026831),pH-sensitive liposomes (see, e.g., U.S. Patent Publication No.2002/0192274; and AU 2003/210303), antibody-coated liposomes (see, e.g.,U.S. Patent Publication No. 2003/0108597; and PCT Publication No. WO00/50008), cell-type specific to liposomes (see, e.g., U.S. PatentPublication No. 2003/0198664), liposomes containing nucleic acid andpeptides (see, e.g., U.S. Pat. No. 6,207,456), liposomes containinglipids derivatized with releasable hydrophilic polymers (see, e.g., U.S.Patent Publication No. 2003/0031704), lipid-entrapped nucleic acid (see,e.g., PCT Publication Nos. WO 03/057190 and WO 03/059322),lipid-encapsulated nucleic acid (see, e.g., U.S. Patent Publication No.2003/0129221; and U.S. Pat. No. 5,756,122), other liposomal compositions(see, e.g., U.S. Patent Publication Nos. 2003/0035829 and 2003/0072794;and U.S. Pat. No. 6,200,599), stabilized mixtures of liposomes andemulsions (see, e.g., EP1304160), emulsion compositions (see, e.g., U.S.Pat. No. 6,747,014), and nucleic acid micro-emulsions (see, e.g., U.S.Patent Publication No. 2005/0037086).

If suitable, any of the agents of the disclosure, includingpharmaceutical drugs, biologics, and therapeutic antibodies, may also bedelivered via the above described carrier systems. All carrier systemsmay further be modified with a targeting moiety or the like in order tofacilitate delivery of the composition to a site of infection in therespiratory airways.

It will be appreciated that conventional means for delivering activeagents are often severely limited by biological, chemical, and physicalbarriers. Typically, these barriers are imposed by the environmentthrough which delivery occurs, the environment of the target fordelivery, or the target itself. Biologically or chemically active agentsare particularly vulnerable to such barriers. In the delivery to animalsof biologically active or chemically active pharmacological andtherapeutic agents, physical and chemical barriers are imposed by thebody. Examples of physical bathers are the skin and various organmembranes that are traversed before reaching a target, and examples ofchemical barriers include, but are not limited to, variations in pH,lipid bilayers, and degrading enzymes. The cellular membrane alsorepresents an important barrier having a significant effect on theeffectiveness of drug delivery.

Immunotherapeutic Agents

In another aspect, the disclosure employs one or more immunotherapeuticagents that may further enhance the respiratory infection clearingeffects imparted by the use of the antimicrobial/antiviral therapeuticagent. For example, the immunotherapeutic agent may be delivered afterthe effects of the antimicrobial agent has set in, but the disclosure isnot limited to this concept. The disclosure contemplates anyadministration regimen involving multiple agents so long as thetherapeutic benefits attributable to each of the agents may occur. It isalso contemplated within the scope of the disclosure that administrationof the one or more immunotherapeutic agents may have immunostimulatoryactivity that provides prophylaxis against further recurrence of aninfection. This immunostimulatory effect may be achieved when the agentis given intranasally or systemically.

Those skilled in the art will appreciate that an immunotherapeutic agentis a treatment that aims to use an individual's own immune system tofight infection or disease. This may be accomplished by boosting theindividual's own immune system or to provide supplemental pieces of anotherwise defective or deficient immune system.

Immunotherapy is a form of biological therapy that can be used in thepresent disclosure to supplement and/or enhance the effects of treatingwith the therapeutic agent. There are generally two recognized forms ofimmunotherapy, which are referred to as active immunotherapies andpassive immunotherapies. Active immunotherapies stimulate the body's ownimmune system to fight a disease. Passive immunotherapies use immunesystem components, such as antibodies, prepared outside the body, toenhance the body's immune response level. Immunotherapies may also workby targeting certain types of cells or antigens (specificimmunotherapies) or they may work by more generally stimulating theimmune system (non-specific immunotherapies, or sometimes referred to asadjuvants). Some examples of immunotherapies contemplated by thedisclosure include monoclonal antibody therapy, non-specificimmunotherapies and adjuvants (substances which boost the immuneresponse such as interleukin-2 and interferon-alpha), immunomodulatingdrugs (such as thalidomide and lenalidomide), and vaccines.

Accordingly, immunotherapeutic agents, which may also be referred to as“immunomodulators” may include, for example, interleukins (e.g., IL-2,IL-7, or IL-12), certain other cytokines (e.g., interferons, growthcolony stimulating factor (G-CSF), imiquimod), chemokines, and othertypes of agents, which can include antigens, epitopes, antibodies,monoclonal antibodies, or even a delivery vehicle to deliver one or moreof these compounds, and may even also include recombinant immune systemcells. Such immunotherapeutic agents may include recombinant forms,synthetic forms, and natural preparations (see D'Alessandro, N. et al.,Cancer Therapy: Differentiation, Immunomodulation and Angiogenesis, NewYork: Springer-Verlag, 1993).

In another embodiment, the immunotherapeutic agent takes advantage ofthe body's innate immune system and has the effect when introduced oftriggering the innate immune response against the unwanted pathogens.

Introduction of the immunotherapeutic agents of the disclosure, may beachieved using any suitable approach, including by local or regionaladministration of the agent at, near, or within the respiratoryinfection. The agent may also be delivered, where suitable, via genetherapy. For example, the antibody-inducing antigen may be introduced byinjecting or otherwise directly administering a genetic vector orotherwise nucleic acid molecule capable of expressing the desiredantigen. The antigens themselves may also be directly administered intothe target infected tissue.

Target Respiratory Diseases

The present disclosure contemplates treating a broad range ofrespiratory diseases, including infections of all types, locations,sizes, and characteristics. For example, the methods of the disclosureare suitable for treating, for example, sinusitis, influenza, andrhinovirus (the common cold).

In other embodiments, virtually any type of respiratory-relatedinfection may be treated by the present disclosure including, but notlimited to, the following respiratory infections: tonsillitis,pharyngitis, laryngitis, sinusitis, otitis media, certain types ofinfluenza, bronchitis, pneumonia, and the common cold.

The compositions of the invention are contemplated to be suitable forprophylaxis and/or treatment of infection from any serotype of humanrhinovirus (HRV). HRV may include, without limitation, the speciesRhinovirus A (including serotypes HRV-A1, HRV-A2, HRV-A7, HRV-A8,HRV-A9, HRV-A10, HRV-A11, HRV-A12, HRV-A13, HRV-A15, HRV-A16, HRV-A18,HRV-A19, HRV-A20, HRV-A21, HRV-A22, HRV-A23, HRV-A24, HRV-A25, HRV-A28,HRV-A29, HRV-A30, HRV-A31, HRV-A32, HRV-A33, HRV-A34, HRV-A36, HRV-A38,HRV-A39, HRV-A40, HRV-A41, HRV-A43, HRV-A44, HRV-A45, HRV-A46, HRV-A47,HRV-A49, HRV-A50, HRV-A51, HRV-A53, HRV-A54, HRV-A55, HRV-A56, HRV-A57,HRV-A58, HRV-A59, HRV-A60, HRV-A61, HRV-A62, HRV-A63, HRV-A64, HRV-A65,HRV-A66, HRV-A67, HRV-A68, HRV-A71, HRV-A73, HRV-A74, HRV-A75, HRV-A76,HRV-A77, HRV-A78, HRV-A80, HRV-A81, HRV-A82, HRV-A85, HRV-A88, HRV-A89,HRV-A90, HRV-A94, HRV-A95, HRV-A96, HRV-A98, HRV-A100, HRV-A101,HRV-A102 and HRV-A103), Rhinovirus B (including the serotypes HRV-B3,HRV-B4, HRV-B5, HRV-B6, HRV-B14, HRV-B17, HRV-B26, HRV-B27, HRV-B35,HRV-B37, HRV-B42, HRV-B48, HRV-B52, HRV-B69, HRV-B70, HRV-B72, HRV-B79,HRV-B83, HRV-B84, HRV-B86, HRV-B91, HRV-B92, HRV-B93, HRV-B97, andHRV-B99), and Rhinovirus C (including, without limitation, serotypesHRV-C1, HRV-C2, HRV-C3, HRV-C4, HRV-C5, HRV-C6, HRV-C7, HRV-C8, HRV-C9,HRV-C10, HRV-C11, HRV-C12, HRV-C13, HRV-C14, HRV-C15, HRV-C16, HRV-C17,HRV-C18, HRV-C19, HRV-C20, HRV-C21, HRV-C22, HRV-C23, HRV-C24, HRV-C25,HRV-C26, HRV-C27, HRV-C28, HRV-C29, HRV-C30, HRV-C31, HRV-C32, HRV-C33,HRV-C34, HRV-C35, HRV-C36, HRV-C37, HRV-C38, HRV-C39, HRV-C40, HRV-C41,HRV-C42, HRV-C43, HRV-C44, HRV-C45, HRV-C46, HRV-C47, HRV-C48, HRV-C49,HRV-C50 and HRV-C51). In some embodiments, the inventive compositionsare contemplated to be useful in the prophylaxis or treatment of viralinfection from any rhinovirus or enterovirus, and in particular anyvirus which binds Intercellular adhesion molecule 1 (ICAM-1). Thecompositions of the invention are also contemplated to be suitable forprophylaxis and/or treatment of infection from any serotype of humaninfluenza virus, including without limitation, those of the generaInfluenzavirus A, Influenzavirus B, and Influenzavirus B, including thespecies influenza A virus (including, without limitation, serotypesH1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, and H7N9 toname a few), influenza B virus, and influenza C virus. In someembodiments, the inventive compositions are contemplated to be useful inthe prophylaxis or treatment of viral infection from any sialicacid-binding virus, including influenza virus, reovirus, adenovirusand/or rotavirus. When sprayed into the nasal cavity and/or mouth, thecompositions of the invention form a deposit on the mucosa, ideallyhaving a long residence time (e.g., at least 1 minute, at least 5minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes,at least 25 minutes, or at least 30 minutes) on the mucosa, butdesirably do not cause excessive drying or irritation of the mucosa.Preferred compositions according to the invention are applied to thenasal and/or oral mucosa for prophylaxis or treatment of humanrhinovirus and human influenza virus infection.

The present disclosure may generally treat and/or prevent all forms ofthe above infections. For example, the method of the disclosureadvantageously may treat or prevent infections arising in any part ofthe respiratory tract including, but not limited to, the upperrespiratory tract (nose, sinuses, larynx and pharynx) and the lowerrespiratory tract (trachea, primary bronchi, bronchial tubes,bronchioles, and lungs).

Reduction of infection means a measurable decrease in growth of theinfection. For example, and without limitation, the infection may bereduced by at least about a factor of 10 (for example 100, 1000-fold ormore) or by decrease of at least about 10% (for example at least about20, 30, 40, 50, 60, 70, 80, 90, 95, 99 or 100%) as compared to thegrowth measured over time prior to treatment as defined herein. Thereduction in infection according to the invention is ideally of astatistically significant degree as compared to otherwise identicalinfected tissues in the absence of the active ingredients contained thecomposition of the invention.

Full eradication of the infection may also be achieved through methodsof the disclosure. Eradication refers elimination of the infection andinfectious organisms. The infection is considered to be eliminated whenit is no longer detectable using detection methods known in the art.

Pharmaceutical Compositions

The disclosure provides pharmaceutical compositions for use in any ofthe methods described herein. The pharmaceutical compositions maycontain a antimicrobial/antiviral therapeutic agent and optionally, animmunotherapeutic agent.

In embodiments, the pharmaceutical compositions include apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the therapeutic is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil, olive oil, gel (e.g., hydrogel),castor oil, and the like. Saline is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions may also beemployed as liquid carriers, particularly for injectable solutions.

The pharmaceutically acceptable carrier may be selected to provide aspecified residence time in the mucosa of a subject. In someembodiments, the “residence time” of the inventive compositions on themucosa represent average residence times from studies involving multipleapplications (intranasal and/or oral) using a sample of multipleindividuals sufficient to approximate the population at large. In someembodiments, at least 25% (and preferably, at least 30%, or at least 40%or at least 50%, or at least 60%, or at least 70%, or at least 80%, orat least 90%) by weight of the initially applied active ingredientsremain on the mucosa after the specified duration of time. In someembodiments, the pharmaceutically acceptable carrier at 25° C. has theHansen Solubility Parameters of energy from dispersion (δ_(a)), energyfrom dipolar intermolecular force between molecules (δ_(p)), energy fromhydrogen bonds (δ_(h)) of between about 15 and about 18, about 12 andabout 15, about 21 and about 25, respectively.

The pharmaceutically acceptable carrier may be aqueous. In someembodiments, the pharmaceutically acceptable carrier is free ofmercurial preservatives. The solvent may be 1,2-propanediol,1,3-propanediol and a variety of aqueous carriers can be used, e.g.buffered water, 0.9 percent saline, buffered aqueous-ethanol solutionsand the like. Combinations of any of these carriers are within the scopeof the invention. These compositions can be sterilized by conventional,well-known sterilization techniques, or can be sterile filtered. Theresulting solutions can be packaged for use as is or mixed as anadjuvant to another medication. A composition can containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, taste modifiers, sweeteners, wettingagents and the like, for example, sodium acetate, sodium lactate, sodiumchloride, potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, etc. In some embodiments, the pharmaceuticallyacceptable carrier is a mixture of water and a polyol. In someembodiments, the pharmaceutically acceptable carrier is a mixture ofwater and propanediol (e.g. 1,2-propendediol, 1,3-propanediol). In someembodiments, the pharmaceutical composition is a mixture of water andglycerin. The pharmaceutically acceptable carrier may be about 1%-about35% (e.g. about 5%-about 30%, etc.) aqueous solution of propanediol orglycerin by weight of the aqueous carrier. Some pharmaceuticallyacceptable carriers include 20% aqueous solution of 1,3-propanediol, 20%aqueous solution of glycerin, 10% aqueous solution of 1,3-propanediol,10% aqueous solution of glycerin, 20% aqueous solution of1,3-propanediol with 1% sunflower oil and 5% polysorbate 80, 20% aqueoussolution of glycerin with 1% sunflower oil and 5% polysorbate 80, 10%aqueous solution of 1,3-propanediol with 1% sunflower oil and 5%polysorbate 80, 10% aqueous solution of glycerin with 1% sunflower oiland 5% polysorbate 80, the Versaflex V-175 polymeric emulsifier system(i.e. sucrose palmitate, glyceryl stearate, glyceryl sterate citrate,sucrose, mannan, and Xanthan gum), the Versaflex V-175 polymericemulsifier system with 3% sunflower oil, the Versaflex V-175 polymericemulsifier system with 3% sunflower oil and about 5 to about 30%propanediol or glycerin, the Versaflex V-175 emulsifier system with 3%acetylated monoglyceride, and the Versaflex V-175 emulsifier system with3% acetylated monoglyceride and about 5 to about 30% propanediol orglycerin.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol, and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. Oral formulation caninclude standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin,the contents of which are hereby incorporated by reference in itsentirety. Such compositions will generally contain a therapeuticallyeffective amount of the therapeutic agent and/or the immunotherapeuticagent, in purified form, together with a suitable amount of carrier soas to provide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In embodiments, the therapeutic agent and/or the immunotherapeutic agentare administered locally as an immediate release or controlled releasecomposition, for example by controlled dissolution and/or the diffusionof the active substance. Dissolution or diffusion controlled release canbe achieved by incorporating the active substance into an appropriatematrix. A controlled release matrix may include one or more of shellac,beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glycerylmonostearate, glyceryl distearate, glycerol palmitostearate,ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetatebutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycols. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated metylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

In related embodiments, the controlled release matrix is a hydrogel. Ahydrogel is a three-dimensional, hydrophilic or amphiphilic polymericnetwork capable of taking up large quantities of water. The networks arecomposed of homopolymers or copolymers, which are insoluble due to thepresence of covalent chemical or physical (e.g., ionic, hydrophobicinteractions, entanglements) crosslinks. The crosslinks provide thenetwork structure and physical integrity. Hydrogels exhibit athermodynamic compatibility with water that allows them to swell inaqueous media. The chains of the network are connected in such a fashionthat pores exist and that a substantial fraction of these pores are ofdimensions between 1 nm and 1000 nm.

The hydrogels can be prepared by crosslinking hydrophilic biopolymers orsynthetic polymers. Examples of the hydrogels formed from physical orchemical crosslinking of hydrophilic biopolymers, include but are notlimited to, hyaluronans, chitosans, alginates, collagen, dextran,pectin, carrageenan, polylysine, gelatin, agarose,(meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate,poly(ethylene glycol) (PEO), poly(propylene glycol) (PPO), PEO-PPO-PEOcopolymers (Pluronics), poly(phosphazene), poly(methacrylates),poly(N-vinylpyrrolidone), PL(G)A-PEO-PL(G)A copolymers, poly(ethyleneimine), and the like. See Hennink and van Nostrum, Adv. Drug Del. Rev.54:13-36 (2002); Hoffman, Adv. Drug Del. Rev. 43:3-12 (2002); Cadee etal., J Control. Release 78:1-13 (2002); Surini et al., J. Control.Release 90:291-301 (2003); and U.S. Pat. No. 7,968,085, each of which isincorporated by reference in its entirety. These materials consist ofhigh-molecular weight backbone chains made of linear or branchedpolysaccharides or polypeptides.

The amount of the pharmaceutical composition of the disclosure that willbe effective in the treatment or prevention of a respiratory infectionor allergy may depend on the nature of the pathogen and can bedetermined by standard clinical techniques, including blood tests and/orimaging techniques. In addition, in vitro assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation may also depend on the route ofadministration, and the seriousness of the infection, and should bedecided according to the judgment of the practitioner and each patient'scircumstances. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

Dosages and Administration Regimens

The therapeutic agents, immunotherapeutic agents, or compositionscontaining these agents are administered in a manner compatible with thedosage formulation, and in such amount as may be therapeuticallyaffective, protective and immunogenic.

The agents and/or compositions may be administered through differentroutes, including, but not limited to, nasal, aerosol, topical, buccaland sublingual, oral, intradermal, subcutaneous, and parenteral. Theterm parenteral as used herein includes, for example, intraocular,subcutaneous, intraperitoneal, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrastemal, intrathecal, intralesional, and intracranial injection, orother infusion techniques.

In some embodiments, administration of the therapeutic agents isdelivered locally or regionally (e.g., intranasally). In someembodiments, a device is used to deliver the antimicrobial compositionto the respiratory tract. The composition may be delivered through useof an inhaler, atomizer, nebulizer, nasal spray bottle, nasal spraypump, ventilator, compressed air tank, aerosolizer, and nasal cannula.The composition can be delivered through insufflation, inhalation, oralingestion, sublingual, and any combination thereof.

In embodiments, the agents and/or compositions formulated according tothe present disclosure are formulated and delivered in a manner to evokea systemic immune response. Thus, in some embodiments, the formulationsare prepared by uniformly and intimately bringing into association theactive ingredient with liquid carriers. Formulations suitable foradministration include aqueous and non-aqueous sterile solutions, whichmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, water, immediately prior to use. Extemporaneous solutions andsuspensions may be prepared from sterile powders, granules and tabletscommonly used by one of ordinary skill in the art.

The agents and/or compositions may be administered in different forms,including, but not limited to, gases, solutions, emulsions andsuspensions, gels, foams, sprays, mists, lotions, microspheres,particles, microparticles, nanoparticles, liposomes, and the like.

The agents and/or compositions are administered in a manner compatiblewith the dosage formulation, and in such amount as may betherapeutically effective, immunogenic and protective. The quantity tobe administered depends on the subject to be treated, including, forexample, the size of the infection and the capacity of the individual'simmune system to synthesize antibodies and/or to produce a cell-mediatedimmune response. Precise amounts of active ingredients required to beadministered depend on the judgment of the practitioner. However,suitable dosage ranges are readily determinable by one skilled in theart and may be of the order of micrograms to milligrams of the activeingredient(s) per dose. The dosage may also depend on the route ofadministration and may vary according to the size of the host.

The agents and/or compositions should be administered to a subject in anamount effective to stimulate a protective immune response in thesubject. Specific dosage and treatment regimens for any particularsubject may depend upon a variety of factors, including the activity ofthe specific compound employed, the age, body weight, general healthstatus, sex, diet, time of administration, rate of excretion, drugcombination, the severity and course of the infection, condition orsymptoms, the subject's disposition to the disease, condition orsymptoms, method of administration, and the judgment of the treatingphysician. Actual dosages can be readily determined by one of ordinaryskill in the art.

Exemplary unit dosage formulations are those containing a dose or unit,or an appropriate fraction thereof, of the administered ingredient. Itshould be understood that in addition to the ingredients mentionedherein, the formulations of the present disclosure may include otheragents commonly used by one of ordinary skill in the art.

Typically in conventional systemically administered treatments, atherapeutically effective dosage should produce a serum concentration ofcompound of from about 0.1 ng/ml to about 50-100 μg/ml. Thepharmaceutical compositions typically provide a dosage of from about0.001 mg to about 2000 mg of compound per kilogram of body weight perday. For example, dosages for administration to a human patient canrange from 1-10 μg/kg, 20-80 μg/kg, 5-50 g/kg, 75-150 μg/kg, 100-500ag/kg, 250-750 ag/kg, 500-1000 ag/kg, 1-10 mg/kg, 5-50 mg/kg, 25-75mg/kg, 50-100 mg/kg, 100-250 mg/kg, 50-100 mg/kg, 250-500 mg/kg, 500-750mg/kg, 750-1000 mg/kg, 1000-1500 mg/kg, 1500-2000 mg/kg, 5 mg/kg, 20mg/kg, 50 mg/kg, 100 mg/kg, 500 mg/kg, 1000 mg/kg, 1500 mg/kg, or 2000mg/kg. Pharmaceutical dosage unit forms are prepared to provide fromabout 1 mg to about 5000 mg, for example from about 100 to about 2500 mgof the compound or a combination of essential ingredients per dosageunit form.

In general, a therapeutically effective amount of the present compoundsin dosage form usually ranges from slightly less than about 0.025mg/kg/day to about 2.5 g/kg/day, preferably about 0.1 mg/kg/day to about100 mg/kg/day of the patient or considerably more, depending upon thecompound used, the condition or infection treated and the route ofadministration, although exceptions to this dosage range may becontemplated by the present disclosure. In an exemplary embodiment,antimicrobial/antiviral compositions according to the present disclosuremay be administered intranasally in amounts ranging from about 0.5 mg/mlof dosing solution to about 50 mg/ml. In another exemplary embodiment,antimicrobial compositions according to the present disclosure may beadministered intranasally in amounts ranging from about 10 mg/ml toabout 30 mg/ml. The dosage of the antimicrobial composition(s) maydepend on the type of infection being treated, the particular compoundused, the therapeutic agent, and other clinical factors and conditionsof the patient. It is to be understood that the present disclosure hasapplication for both human and veterinary use.

The agents and/or compositions may be administered in one or more dosesas required to achieve the desired effect. Thus, the agents and/orcompositions may be administered in 1, 2, to 3, 4, 5, or more doses.Further, the doses may be separated by any period of time, for examplehours, days, weeks, months, and years.

The agents and/or compositions may be formulated as liquids or drypowders, or in the form of microspheres.

The agents and/or compositions may be stored at temperatures of fromabout −100° C. to about 25° C. depending on the duration of storage. Theagents and/or compositions may also be stored in a lyophilized state atdifferent temperatures including room temperature. The agents and/orcompositions may be sterilized through conventional means known to oneof ordinary skill in the art. Such means include, but are not limitedto, filtration.

The amount of active ingredient that may be combined with carriermaterials to produce a single dosage form may vary depending upon thehost treated and the particular mode of administration. In embodiments,a preparation may contain from about 0.1% to about 95% active compound(w/w), from about 20% to about 80% active compound, or from anypercentage there between.

In embodiments, the pH of the formulation may be adjusted withpharmaceutically acceptable acids, bases, or buffers to enhance thestability of the formulated compound or its delivery form.

In embodiments, the pharmaceutical carriers may be in the form of asterile liquid preparation, for example, as a sterile aqueous oroleaginous suspension.

Among the acceptable vehicles and solvents that may be employed aremannitol, water, Ringer's solution and isotonic sodium chloridesolution.

In addition, sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose, any bland fixed oil maybe employed including synthetic mono- or to diglycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, or carboxymethylcellulose or similar dispersing agents which are commonly used in theformulation of pharmaceutically acceptable dosage forms such asemulsions and or suspensions.

Other commonly used surfactants such as TWEEN® or SPAN® and/or othersimilar emulsifying agents or bioavailability enhancers which arecommonly used in the manufacture of pharmaceutically acceptable solid,liquid, or other dosage forms may also be used for the purposes offormulation.

In embodiments, the agents and/or compositions can be delivered in anexosomal delivery system. Exosomes are small membrane vesicles that arereleased into the extracellular environment during fusion ofmultivesicular bodies with plasma membrane. Exosomes are secreted byvarious cell types including hematopoietic cells, normal epithelialcells and even some tumor cells. Exosomes are known to carry MHC classI, various costimulatory molecules and some tetraspanins. Recent studieshave shown the potential of using native exosomes as immunologicstimulants.

Also contemplated by the disclosure is delivery of the agents and/orcompositions using nanoparticles. For example, the agents and/orcompositions provided herein can contain nanoparticles having at leastone or more agents linked thereto, e.g., linked to the surface of thenanoparticle. A composition typically includes many nanoparticles witheach nanoparticle having at least one or more agents linked thereto.Nanoparticles can be colloidal metals. A colloidal metal includes anywater-insoluble metal particle or metallic compound dispersed in liquidwater. Typically, a colloid metal is a suspension of metal particles inaqueous solution. Any metal that can be made in colloidal form can beused, including gold, silver, copper, nickel, aluminum, zinc, calcium,platinum, palladium, and iron. In some cases, gold nanoparticles areused, e.g., prepared from HAuCl₄. Nanoparticles can be any shape and canrange in size from about 1 nm to about 10 nm in size, e.g., about 2 nmto about 8 nm, about 4 to about 6 nm, or about 5 nm in size. Methods formaking colloidal metal nanoparticles, including gold colloidalnanoparticles from HAuCl₄, are known to those having ordinary skill inthe art. For example, the methods described herein as well as thosedescribed elsewhere (e.g., US Pat. Publication Nos. 2001/005581;2003/0118657; and 2003/0053983, which are hereby incorporated byreference) are useful guidance to make nanoparticles.

In certain cases, a nanoparticle can have two, three, four, five, six,or more active agents linked to its surface. Typically, many moleculesof active agents are linked to the surface of the nanoparticle at manylocations. Accordingly, when a nanoparticle is described as having, forexample, two active agents linked to it, the nanoparticle has two activeagents, each having its own unique molecular structure, linked to itssurface. In some cases, one molecule of an active agent can be linked tothe nanoparticle via a single attachment site or via multiple attachmentsites. An active agent can be linked directly or indirectly to ananoparticle surface. For example, the active agent can be linkeddirectly to the surface of a nanoparticle or indirectly through anintervening linker.

Any type of molecule can be used as a linker. For example, a linker canbe an aliphatic chain including at least two carbon atoms (e.g., 3, 4,5, 6, 7, 8, 9, 10 or more carbon atoms), and can be substituted with oneor more functional groups including ketone, ether, ester, amide,alcohol, amine, urea, thiourea, sulfoxide, sulfone, sulfonamide, anddisulfide to functionalities. In cases where the nanoparticle includesgold, a linker can be any thiol-containing molecule. Reaction of a thiolgroup with the gold results in a covalent sulfide (S) bond. Linkerdesign and synthesis are well known in the art.

In embodiments, the nanoparticle is linked to a targeting agent/moiety.A targeting functionality can allow nanoparticles to accumulate at thetarget (e.g. nasal membrane) at higher concentrations than in othertissues. In general, a targeting molecule can be one member of a bindingpair that exhibits affinity and specificity for a second member of abinding pair. For example, an antibody or antibody fragment therapeuticagent can target a nanoparticle to a particular region or molecule ofthe body (e.g., the region or molecule for which the antibody isspecific) while also performing a therapeutic function. In some cases, areceptor or receptor fragment can target a nanoparticle to a particularregion of the body, e.g., the location of its binding pair member. Othertherapeutic agents such as small molecules can similarly target ananoparticle to a receptor, protein, or other binding site havingaffinity for the therapeutic agent.

When the compositions of this disclosure comprise one or more additionaltherapeutic or prophylactic agents, thetherapeutic/enhancing/immunotherapy agent and the additional agentshould be present at dosage levels of between about 0.1 to 100%, orbetween about 5 to 95% of the dosage normally administered in amonotherapy regimen. The additional agents may be administeredseparately, as part of a multiple dose regimen, from the agents of thisdisclosure. Alternatively, those additional agents may be part of asingle dosage form, mixed together with the agents of this disclosure ina single composition.

The administration of the agents and/or compositions of the disclosureelicits an immune response against a pathogen. Typically, the dose canbe adjusted within this range based on, e.g., the subject's age, thesubject's health and physical condition, the capacity of the subject'simmune system to produce an immune response, the subject's body weight,the subject's sex, diet, time of administration, the degree ofprotection desired, and other clinical factors. Those in the art canalso readily address parameters such as biological half-life,bioavailability, route of administration, and toxicity when formulatingthe agents and/or compositions of the disclosure.

The following examples further demonstrate several embodiments of thisdisclosure. While the examples illustrate the disclosure, they are notintended to limit it.

EXAMPLES

The structures, materials, compositions, and methods described hereinare intended to be representative examples of the disclosure, and itwill be understood that the scope of the disclosure is not limited bythe scope of the examples. Those skilled in the art will recognize thatthe disclosure may be practiced with variations on the disclosedstructures, materials, compositions and methods, and such variations areregarded as within the ambit of the disclosure.

Example 1: Administration of Antimicrobial Compositions in Non-HumanSubjects to Prevent Infection

Varying concentrations of antimicrobial compositions containingingredients from Table 1 are administered intranasally to a group ofhealthy, uninfected mice selected for age, gender and weight. After asuitable period of time to allow the compositions to take effect, miceare inoculated nasally with varying doses of respiratory pathogens(influenza, rhinovirus, bacteria, and fungi). At different subsequenttime points, samples are extracted from the mice and analyzed formicrobial infection. Lack of infection indicates the antimicrobialcomposition prevents the airborne pathogens from infecting the mice. Theantimicrobial composition enhances the filtering capabilities of thenasal membrane and protects against the airborne pathogens.

Example 2: Administration of Antimicrobial Compositions in Non-HumanSubjects to Treat Infection

A group of healthy, uninfected mice selected for age, gender and weightare inoculated nasally with varying doses of respiratory pathogens(influenza, rhinovirus, bacteria, and fungi). After allowing suitabletime for the pathogens to infect the mice, varying concentrations ofantimicrobial compositions containing ingredients from Table 1 (as inExample 1) are administered intranasally to the infected mice. After asuitable period of time to allow the compositions to take effect,samples are extracted from the mice and analyzed for microbialinfection. Lack of infection indicates the antimicrobial compositiontreats the respiratory infections within the mice. The antimicrobialcomposition enhances the filtering capabilities of the nasal membraneand treats the infection caused by airborne pathogens.

Example 3: Administration of Antimicrobial Compositions in HumanSubjects to Treat Infection

A group of human subjects presenting without pre-existing influenza orrhinoviral infections are selected for treatment and their baselineblood drawn to screen for markers of respiratory infection.Antimicrobial compositions containing ingredients from Table 1 (as inExamples 1 and 2) are administered intranasally to the subjects. After asuitable period of time to allow the compositions to take effect,subjects are exposed to airborne rhinovirus or influenza. After asuitable period of time to determine whether they had been infected,their bloods would again be drawn and screened for systemic markers ofrespiratory infection and they would be observed and questioned forvisible evidence of respiratory infection. Lack of infection indicatesthe antimicrobial composition prevents respiratory infections. Theantimicrobial composition enhances the filtering capabilities of thenasal membrane and prevents the viruses from causing respiratoryinfection.

Example 4: Measurements of Compositions on Full Differentiated 3D CellModel of the Human Airway Epithelia Inoculated with Rhinovirus A16

Various compositions were tested for their ability to protect a 3D modelof human airway epithelium, constituted with primary human epithelialcells freshly isolated from nasal, tracheal or bronchial biopsies(MucilAir™). MucilAir™ is composed of basal cells, ciliated cells andmucus cells from the respiratory tract. The proportion of these variouscell types is preserved compared to what one observes in vivo (Huang etal., Drug Discovery and Development-Present and Future, 8, 201).Moreover, the epithelia are started from de-differentiated cells.Epithelia (MucilAir™-Pool) were reconstituted with a mixture of cellsisolated from 14 different normal nasal donors and cultured for 41 days.Epithelial cells were freshly isolated from biopsies (nose and bronchi),then seeded onto a semi-porous membrane (Costar Transwell, pore size 0.4m). After about 45 days of culture at air-liquid interface, theepithelia were fully differentiated, both morphologically andfunctionally. After 45 days of culture, the epithelia are fullyciliated, secreted mucus and are electrically tight (TEER>200 Ω cm²).The activity of the main epithelial ionic channels, such as CFTR, EnaC,Na/K ATPase, is preserved and the epithelia is shown to respond in aregulated and vectorial manner to the pro-inflammatory stimulus, TNF-α(Huang et al., 2011 and Huang et al., 3R-Info-Bulletin No. 41, October2009).

Compositions with various active ingredients were prepared as shown inTable 5. Each composition was prepared in a buffered saline solution(0.9% NaCl, 1.25 mM CaCl₂, 10 mM HEPES). As used herein, “HRV1” refersto compositions comprising apolactoferrin (i.e., HRV1-1, HRV1-2, andHRV1-3) as the sole active, “HRV2” refers to compositions comprisinglysozyme (i.e., HRV2-1, HRV2-2, and HRV2-3) as the sole active, andreference to “HRV3” refers to compositions comprising soluble ICAM-1(sICAM) (i.e., HRV3-1, HRV3-2, and HRV2-3) as the sole active. Referenceto “HRV4” refers to compositions comprising a combination ofapolactoferrin, lysozyme and soluble ICAM-1.

TABLE 5 Composition Name Active Ingredients (Concentration) HRV1-1Apolactoferrin (500 μg/mL) HRV1-2 Apolactoferrin (50 μg/mL) HRV1-3Apolactoferrin (5 μg/mL) HRV2-1 Lysozyme (2500 μg/mL) HRV2-2 Lysozyme(250 μg/mL) HRV2-3 Lysozyme (25 μg/mL) HRV3-1 soluble ICAM-1 (50 μg/mL)HRV3-2 soluble ICAM-1 (5 μg/mL) HRV3-3 soluble ICAM-1 (0.5 μg/mL) HRV4-1Apolactoferrin (500 μg/mL) Lysozyme (2500 μg/mL) soluble ICAM-1 (50μg/mL) HRV4-2 Apolactoferrin (50 μg/mL) Lysozyme (250 μg/mL) solubleICAM-1 (5 μg/mL) HRV4-3 Apolactoferrin (5 μg/mL) Lysozyme (25 μg/mL)soluble ICAM-1 (0.5 μg/mL)

20 μL of each formulation was applied apically onto separateMucilAir™-Pools immediately prior to inoculation (time=0) with HumanRhinovirus-A16. 20 μL of the each of the formulations was also appliedat 3.5 and 24 hours post-inoculation (“pi”). Innoculation with HumanRhinovirus-A16 was achieved by the application of 50 μL of 2×10⁶/mlHuman Rhinovirus A16 (clinical strain: QCHRV.16) on the apical side ofthe 3D model for 3 h at 34° C., 5% CO₂. The virus stocks were producedin MucilAir™ cultures and diluted in culture medium without purificationor concentration.

After inoculation (time=0), epithelia were washed twice with MucilAir™culture medium in order to clean the inoculum. Cell free, apical washes(20 minutes) with 200 MucilAir™ culture media were collected at 3.5hours post-inoculation and then at 24 and 48 hours pi and stocked at−80° C.

From the apical washes, viral RNA was extracted with the QIAamp® ViralRNA kit (Qiagen). Viral RNA was quantified by quantitative RT-PCR(QuantiTect Probe RT-PCR, Qiagen) with the TaqMan ABI 7000. Using knownconcentration of the corresponding viral RNA to establish a standardcurve, the quantification was absolute. Data are presented as genomecopy number/ml on the graphs illustrating the results of viralreplication, unless otherwise indicated. Experiments and data with a“(+)” designation refer to experiments performed on inoculated media andexperiments and data with a “(−)” designation refer to experimentsperformed on media not inoculated with virus. To compare two sets ofdata, Students unpaired t-test was used. To compare means of three ormore samples, One-way analysis of variance (ANOVA) was performed withDunnett's multiple comparison tests (***=p<0.001, **=p<0.01, *=p<0.05).As negative control, non-infected and non-treated cultures (Mock) wereused.

To compare the potential effects of HRV compounds, positive controlswere included. For the toxic effect, cultures were treated with 20 μL of10% Triton X-100 in a buffered saline solution (0.9% NaCl, 1.25 mMCaCl₂), 10 mM HEPES). For the anti-Rhinovirus effect, 20 μL of 5 MRupintrivir was added to basal medium. Rupintrivir (Santa CruzBiotechnologies) stock solution of 2 mM in DMSO (−20° C.) was diluted to5 μM in MucilAir™ medium (0.25% DMSO final concentration).

Error bars in any figure refer to Standard Error of the Mean (SEM). Allcomparisons are versus the data from vehicle infected (without active)and all data reported are single measurement on three separate inserts(n=3). All reported results are statistically significant.

TEER Measurements

Tissue integrity was monitored using transepithelial electricalresistance (“TEER”) measurements. TEER is a dynamic parameter thatreflects the state of epithelia that can be affected by several factors.For example, if holes were present or if cellular junction were broken,the TEER values would be generally below 100 Ω cm². In contrast, whenepithelia are not damaged, the TEER values are typically above 200 Ωcm². A notable decrease of the TEER values (but >100 Ω cm²) generallyreflects an activation of the ion channels. A drastic increase of theTEER value reflects a blockage of the ion channel activity or adestruction of the ciliated cells. When an epithelium is damaged, adecrease of TEER would be associated with an increase of LDH release ora decrease of the cell viability. TEER monitoring was performed 24 (D1)and 48 (D2) hours post-inoculation. The Triton X-100 control correspondsto a loss of TEER (<100 Ω cm²) after cell damage. After addition of 200μL of MucilAir™ medium to the apical compartment of the MucilAir™cultures, resistance was measured with an EVOMX volt-ohm-meter (WorldPrecision Instruments UK, Stevenage) for each condition. Measuredresistance values (Q) were converted to TEER (Ω cm²) with the membraneresistance (100Ω) connected in series to the epithelium. The epitheliumhas a total surface area of 0.33 cm². TEER may be calculated by thefollowing formula:

TEER (Ωcm²)=(resistance value(Ω)−100(Ω))×0.33 (cm²)

The results of TEER measurements are found in FIGS. 1-4. As can be seen,no significant change in TEER was observed at 24 (D1) hours pi or at 48(D2) hours pi for any of the HRV tes formulations.

Lactate Dehydrogenase Release

Lactate dehydrogenase (“LDH”) is a stable cytoplasmic enzyme that israpidly released into the culture medium upon rupture of the plasmamembrane. 100 basolateral medium collected at each time-point wasincubated with the reaction mixture of the Cytotoxicity DetectionKitPLUS, following manufacturer's instructions (Sigma, Roche,11644793001). The amount of the released LDH was then quantified bymeasuring the absorbance of each sample at 490 nm with a microplatereader. To determine the percentage of cytotoxicity, the difference ofexperimental absorbance (A_(exp)) from a low control is compared to thedifference between Absorbance values of the low (A_(low)) and highcontrol (A_(high)), using the following equation:

Cytotoxicity (%)=(A _(exp) −A _(low))/(A _(high) −A _(low))

A percentage below 5% reflects a physiological release of LDH in themedium. LDH measurements were taken at 24 and 48 hours pi. The resultsare shown in FIGS. 5-8. As can be seen, drug formulations do notincrease LDH release in the 3D models.

Cilia Beating Frequency

Cilia beating frequency (“CBF”) was measured by an experimental systemconsisting of three parts: a camera (Sony XCD V60 Firewire), a PCI cardand a specific package of software. 256 images were captured at highfrequency rate (125 fps) at room temperature and the cilia beatingfrequency was then calculated using Epithelix software. CBF values maybe subject to fluctuations due to parameters such as temperature, mucusviscosity or liquid (such as a buffered saline solution) applied on theapical surface of the MucilAir™ 3D epithelial model. Therefore resultsare considered significant when a ratio >20% between the infectedvehicle control and the drug composition was reached. FIGS. 9-12illustrate the results of the cilia beating frequency measurements taken24 and 48 hours pi. As can be seen, HRV treatments showed no significanteffect on cilia beating frequency.

Mucociliary Clearance

The mucociliary clearance (“MCC”) was monitored using a Sony XCD-U100CRcamera connected to an Olympus BX51 microscope with a 5× objective.Polystyrene microbeads of 30 m diameter (Sigma, 84135) were added on theapical surface of MucilAir™. Microbead movements were video tracked at 2frames per second for 30 images at room temperature. Three movies weretaken per insert. Average beads movement velocity (Ωm/sec) wascalculated with ImageProPlus 6.0 software. Mucociliary clearance valuesless than 10 μm/s are considered pathological. FIG. 13 illustrates theeffect of Rhinovirus A16 infection on MCC on treatment with each of theactives alone and in combination measured 48 hours pi. As can be seen,combination treatment demonstrated superior and consistent responseacross the doses tested, as compared to the other test formulations. MCCwas decreased for lower concentrations of HRV1 formulations and theHRV3-3 formulation. However, these negative effects were not seen withthe HRV4 formulations at any concentration. Surprisingly, even thoughapolactoferring alone inhibited ciliary movement at some doses (see,e.g., HRV1-1 and HRV1-2), the effect was completely ameliorated in theHRV combination at each dose.

Apical Rhinovirus Replication

From the 200 μL apical washes, 20 μL was used for viral RNA extractionwith the QIAamp® Viral RNA kit (Qiagen) resulting in 60 μL RNA elutionvolume. Viral RNA was quantified by quantitative RT-PCR (QuantiTectProbe RT-PCR, Qiagen) using 5 μL of viral RNA, Mastermix, twoPicornaviridae family specific and a Pan-Picornaviridae primers, and aPicornaviridae probe with FAM-TAMRA reporter-quencher dyes. Fourdilutions of known concentration of Rhinovirus A16 as well as controlsfor RNA extraction and RT-PCR were included and the plates were run on aTaqMan ABI 7000 from Applied Biosystems. Count (“Ct”) data were reportedto the standard curve, corrected with the dilutions and presented asgenome copy number/ml. FIGS. 14-17 illustrate the results of RhinovirusA16 replication. As can be seen Rhinovirus showed a significantreplication which was inhibited by Ruptinitrivir. No significant changein Rhinovirus replication was achieved using HRV1 and HRV2 formulations.Application of HRV3 formulations results in a dose response relationshipsimilar to that of Ruptinitrivir. HRV4 formulations show a dosedependent response on Rhinovirus A16 replication too a far greaterdegree than Ruptinitrivir treatment.

Enzyme-Linked Lectin Assay

Mucin secretion was quantified using an Enzyme-linked Lectin Assay(“ELLA”) protocol detecting the carbohydrates groups of the collectedmucus. 96-well plates were coated with 6 g/ml Lectin from Triticumvulgaris (wheat) (Sigma, L0636) in phosphate buffered solution (“PBS”)adjusted at pH 6.8 and incubated for 1 hour at 37° C. After washingsteps with high salt phosphate buffered saline (PBS) (0.5 M NaCl, 0.1%Tween-20 in PBS), samples and standards (Mucin from porcine stomach TypeII, Sigma, M2378) were incubated for 30 minutes at 37° C. After washing,plates were incubated 30 minutes at 37° C. with a detection solutioncontaining 1 g/ml of Peroxidase conjugated Lectin from Glycine max(soybean) (Sigma, L2650), in 0.1% BSA-PBS adjusted at pH 7.4. After thefinal washing steps, a substrate reagent (TMB) was added and incubatedfor 10 minutes in the dark at room temperature. The reaction was stoppedwith 2N H₂SO₄ and the plates were read at 450 nm. FIGS. 18-21 illustratethe mucin quantity from the apical medium at 24 and 48 hours. As can beseen, the HRV test formulations showed no significant effect on mucinsecretion.

Example 5: Measurements of Compositions on Full Differentiated 3D CellModel of the Human Airway Epithelia Inoculated with Influenza a H1N1

Epithelia (MucilAir™-Pool) were reconstituted with a mixture of cellsisolated from 14 different normal nasal donors and cultured for 41 days.Compositions with various active ingredients were prepared as shown inTable 6. Each composition was prepared in a buffered saline solution(0.9% NaCl, 1.25 mM CaCl₂, 10 mM HEPES). As used herein, “IAV1” refersto compositions comprising apolactoferrin (i.e., IAV1-1, IAV 1-2, andIAV 1-3), reference to “IAV2” refers to compositions comprising lysozyme(i.e., IAV 2-1, IAV 2-2, and IAV 2-3), and reference to “IAV3” refers tocompositions comprising soluble ICAM-1 (i.e., IAV 3-1, IAV 3-2, and IAV2-3). Reference to “IAV4” refers to example compositions comprising acombination of apolactoferrin, lysozyme, 3′-sialyllactose, and6′-sialyllactose. These various test formulations are shown below inTable 6.

TABLE 6 Composition Name Active Ingredients (Concentration) IAV1-1Apolactoferrin (500 μg/mL) HRV1-2 Apolactoferrin (50 μg/mL) IAV1-3Apolactoferrin (5 μg/mL) IAV2-1 Lysozyme (2500 μg/mL) IAV2-2 Lysozyme(250 μg/mL) IAV2-3 Lysozyme (25 μg/mL) IAV3-1 3′-sialyllactose (327μg/mL) 6′-sialyllactose (327 μg/mL) IAV3-2 3′-sialyllactose (3.27 μg/mL)6′-sialyllactose (3.27 μg/mL) IAV3-3 3′-sialyllactose (0.327 μg/mL)6′-sialyllactose (0.327 μg/mL) IAV4-1 Apolactoferrin (500 μg/mL)Lysozyme (2500 μg/mL) 3′-sialyllactose (327 μg/mL) 6′-sialyllactose (327μg/mL) IAV4-2 Apolactoferrin (50 μg/mL) Lysozyme (250 μg/mL)3′-sialyllactose (3.27 μg/mL) 6′-sialyllactose (3.27 μg/mL) IAV4-3Apolactoferrin (5 μg/mL) Lysozyme (25 μg/mL) 3′-sialyllactose (0.327μg/mL) 6′-sialyllactose (0.327 μg/mL))

20 μL of each formulation was applied apically onto separateMucilAir™-Pools immediately prior to inoculation with Influenza A H1N1.20 μL of the each formulation was also applied at 3.5 and 24 hourspost-inoculation. Innoculation with Influenza A H1N1 (t=0) was achievedby the application of 50 μL of 2×10⁶/ml H1N1 (clinical strain:A/California/7/09) on the apical side of MucilAir™ tissue for 3 h at 34°C., 50% CO₂. The virus stocks were produced in MucilAir™ cultures anddiluted in culture medium without purification or concentration.Measurements on TEER, LDH release, CBF, MCC and mucin secrection wereperformed in an otherwise identical fashion as described above, exceptthat 10 μM Oseltamivir was used instead of Ruptinivir for the antiviraleffect formulation. For the antiviral effect, 10 μM Oseltamivir wasadded to basal medium. Oseltamivir acid (Carbosynth) stock solution of 4mM in DMSO (−20° C.) was diluted to 10 μM in MucilAir™ basolateralmedium (0.25% DMS final concentration).

TEER Measurements

FIGS. 22-25 illustrate the results of TEER measurements with Influenza AH1N1. As can be seen, the cytopathic effect of the influenza viruscaused a decrease in the TEER resistance measurement. The IAV3 and IAV4test formulations appear to limit the decrease in TEER at 48 hours (D2)pi, with the mitigation of resistance loss most pronounced informulations comprising higher concentrations of actives (i.e., IAV4-2and IAV4-1).

Lactate Dehydrogenase Release

FIGS. 26-29 illustrate the results of LDH release from the epithelialcells. As can be seen, no cytotoxic effect was observed for any of theIAV formulations.

Cilia Beating Frequency

FIGS. 30-33 illustrate the effects of the various treatments on the CBFof epithelial cells with Influenza H1N1 infection. As can be seen, IAV4showed no significant effect on CBF.

Mucociliary Clearance

FIG. 34 illustrates the results on mucociliary clearance after treatmentwith the specified test formulations. As can be seen, the IAVformulations showed no significant effect on mucociliary clearance.

Apical Rhinovirus Replication

From the 200 μL apical washes, 20 μL was used for viral RNA extractionwith the QIAamp® Viral RNA kit (Qiagen) resulting in 60 μL RNA elutionvolume. Viral RNA was quantified by quantitative RT-PCR (QuantiTectProbe RT-PCR, Qiagen) using 5 μL of viral RNA, Mastermix, two InfluenzaA specific primers and Influenza A probe with FAM-BHQ1 reporter-quencherdyes. Four dilutions of known concentration of H1N1 as well as controlsfor RNA extraction and RT-PCR were included and the plates were run on aTaqMan ABI 7000 from Applied Biosystems. Ct data were reported to thestandard curve, corrected with the dilutions and presented as genomecopy number/ml. FIGS. 35-38 illustrate the results of Influenza A H1N1replication in presence of the specified test formulation. As can beseen Influenza showed a significant replication which was inhibited byOseltamivir. No significant change in Influenza A H1N1 replication wasachieved using IAV formulations.

Enzyme-Linked Lectin Assay

FIG. 39-42 illustrate the mucin quantity from the apical medium at 24(D1) and 48 (D2) hours pi. As can be seen, IAV4 showed a dose dependentresponse in mucin secretion.

As shown by TEER measurements, H1N1 infection results in a loss oftissue integrity on human airway epithelia resulting in the breakdown ofthe barrier function of the airway. This results in further infectionand inflammation. A decrease of TEER is the earliest and most sensitiveparameter affected by H1N1 infection. Combination formulations IAV3 andIAV4 resulted in the partial mitigation of this breakdown of tissueintegrity. Additionally, these formulations prevented any of thenegative effects on CBF that was seen in IAV1 and IAV2 formulations.

Having thus described in detail a number of preferred embodiments of thepresent disclosure, it is to be understood that the disclosure definedby the above paragraphs is not to be limited to particular details setforth in the above description, as many apparent variations thereof arepossible without departing from the spirit or scope of the presentdisclosure.

All documents cited or referenced herein and all documents cited orreferenced in the herein cited documents, together with anymanufacturer's instructions, descriptions, product specifications, andproduct sheets for any products mentioned herein or in any documentincorporated by reference herein, are hereby incorporated by reference,and may be employed in the practice of the disclosure.

What is claimed is:
 1. A pharmaceutical composition comprising immunoglobulin G (IgG) and one or more nanoparticles together with one or more pharmaceutically acceptable carriers or excipients, wherein said pharmaceutical composition is formulated for delivery to the nasal membrane of a subject for prophylaxis against viral infections of the respiratory tract.
 2. The pharmaceutical composition according to claim 1, wherein said pharmaceutical composition is formulated as a gas, liquid, spray, gel, foam, mist lotion, syrup, or powder.
 3. The pharmaceutical composition according to claim 1, wherein said pharmaceutical composition is formulated as a nasal spray.
 4. The pharmaceutical composition according to claim 1, wherein the IgG is linked to the surface of the nanoparticle.
 5. The pharmaceutical composition according to claim 1, wherein the IgG is linked directly to the surface of the nanoparticle.
 6. The pharmaceutical composition according to claim 1, wherein the IgG is linked indirectly to the surface of the nanoparticle through an intervening linker.
 7. The pharmaceutical composition according to claim 6, wherein said intervening linker is an aliphatic chain comprising at least two carbon atoms.
 8. The pharmaceutical composition according to claim 7, wherein said intervening linker is an aliphatic chain comprising from 2-10 carbon atoms.
 9. The pharmaceutical composition according to claim 7, wherein said aliphatic chain is substituted with one or more functional groups selected from ketone, ether, ester, amide, alcohol, amine, urea, thiourea, sulfide, sulfoxide, sulfone, sulfonamide, and disulfide.
 10. The pharmaceutical composition according to claim 1, wherein said pharmaceutical composition is formulated to form a deposit on the nasal membrane having a residence time thereon of at least 10 minutes.
 11. The pharmaceutical composition according to claim 1, wherein said pharmaceutically acceptable carrier comprises an organic liquid having Hansen Solubility Parameters of: a) an energy of dispersion (δ_(d)) of between about 15 and about 18; b) an energy from dipolar intermolecular force between molecules (δ_(p)) of between about 12 and about 18; and c) an energy from hydrogen bonds (δ_(h)) of between about 21 and about
 25. 12. The pharmaceutical composition according to claim 1, wherein said composition further comprises one or more ingredients selected from the group consisting of an emollient, an occlusive, a humectant, an emulsifier, and an essential oil.
 13. The pharmaceutical composition according to claim 1, wherein said composition further comprises an isotonicity adjuster.
 14. The pharmaceutical composition according to claim 1, wherein said composition is isotonic to nasal epithelia.
 15. The pharmaceutical composition according to claim 1, wherein said carrier comprises a polyol.
 16. The pharmaceutical composition according to claim 1, wherein said composition has a kinematic viscosity ranging from 1-1500 centiStokes (mm²/s).
 17. A nasal spray comprising immunoglobulin G (IgG) and one or more nanoparticles together with one or more pharmaceutically acceptable carriers or excipients, wherein said nasal spray is formulated for delivery to the nasal membrane of a subject for prophylaxis against viral infections of the respiratory tract, and said nasal spray is isotonic to nasal epithelia.
 18. The nasal spray according to claim 17, wherein said nasal spray readily flows through a spray nozzle and forms a mist of suitable droplet size on shearing for deposition of said composition on said nasal membrane.
 19. A method for the prevention of viral infection in a subject comprising administering the pharmaceutical composition according to claim 1 to the nasal membrane of the subject.
 20. A method for the prevention of viral infection in a subject comprising administering the pharmaceutical composition according to claim 17 to the nasal membrane of the subject. 